Characterisation of Plasmodium Sexual Stage Antigens as Targets of Transmission Blocking Immunity

A promising strategy to achieve malaria elimination is by interrupting transmission using transmission-blocking vaccines (TBVs). TBVs target the sexual or mosquito stages of the malaria parasite to inhibit parasite development within the mosquito. To date, however, there are only three lead TBV candidate antigens Pfs230, Pfs48/45 and Pfs25 in various stages of clinical development. This project sought to identify novel antigens expressed on gametocytes, gametes and ookinetes with potential as TBV candidates. Furthermore, naturally acquired immune responses (NAI) to gametocytes have been described and have the potential to guide the development and the implementation of TBVs Therefore, this work also sought to improve our understanding of NAI to gametocytes. This was achieved by (1) carrying out a systematic review and meta-analysis of studies investigating NAI to the lead gametocyte-stage TBV candidates, and (2) assessing the changing patterns of gametocyte carriage at the Kenyan coast over time. Key indicators of gametocytaemia and anti-gametocyte immunity were identified and evaluated against a novel panel of gametocyte antigens. These antigens, together with a separate set of gamete and ookinete stage antigens, were identified as potential TBV candidates using a combination of bioinformatic tools and laboratory investigations. Immunoprofiling of the identified gametocyte candidates provided evidence that stable responses can be generated to sexual stage antigens. Moreover, antigens that could serve as serological markers of recent gametocyte exposure, in particular PEB-P (PF3D7_0303900), were also identified. The ability of the sexual stage antigens to induce transmission-blocking immunity was also assessed. Promising candidates identified included PBCPP2 (PBANKA_0719100), a novel conserved and uncharacterised P. berghei protein), and SOAP (PBANKA_1037800). Further characterisation of these antigens may yield new candidates to add to the TBV development pipeline

Plasmodium falciparum protein kinase CK2

Malaria, caused by infection with intracellular protozoan parasites of the genus Plasmodium, is responsible for 300 to 600 million clinical cases annually (Snow et al., 2005), resulting in the deaths of up to three million people every year (Breman, 2001, Breman et al., 2004). There is a clear need for further research aimed at identifying novel drug targets (Ridley, 2002). Reversible phosphorylation of proteins is a major regulatory mechanism in most cellular processes, and protein kinases are considered promising drug targets, comprising as much as 30% of all protein targets under investigation (Cohen, 2002). The divergences between human and plasmodial protein kinases suggest that specific inhibition of the latter is an achievable goal (Doerig, 2004, Doerig and Meijer, 2007). This study investigates protein kinase CK2 of Plasmodium falciparum, seeking to establish by reverse genetics and biochemical approaches whether it represents a possible antimalarial drug target. Protein-kinase CK2, formerly known as Casein Kinase II, is a dual-specificity (Serine/Threonine and Tyrosine) protein kinase ubiquitously expressed in eukaryotes. It has over 300 cellular substrates catalogued to date (Meggio and Pinna, 2003). Consistent with its multiple substrates, the enzyme plays a crucial role in many cellular processes, and is essential to viability in yeast and slime mould (Padmanabha et al., 1990, Kikkawa et al., 1992). The human CK2 holoenzyme consists of two catalytic a or a’ subunits and two regulatory b subunits, and recent evidence indicates that the latter interact with several protein kinases in addition to CK2a (reviewed in (Bibby and Litchfield, 2005)), pointing to a likely role in the integration of numerous signalling pathways. A putative CK2a orthologue and two predicted CK2b subunits were identified in the P. falciparum genome (Ward et al., 2004, Anamika et al., 2005). Here we present the biochemical characterisation of the PfCK2a orthologue and both PfCK2b orthologues, and demonstrate by using a reverse genetics approach that each of the three subunits is essential for completion of the erythrocytic asexual cycle of the parasite, thereby validating the enzyme as a possible drug target. Recombinant PfCK2a possesses protein kinase activity, exhibits similar substrate and co-substrate preferences to those of CK2a subunits from other organisms, and interacts with both of the PfCK2b subunits in vitro. PfCK2a is amenable to inhibitor screening, and we report differential susceptibility between the human and P. falciparum CK2a enzymes to a small molecule inhibitor. Taken together, the data indicate that PfCK2a is an attractive, validated target for antimalarial chemotherapeutic intervention

Dianas terapéuticas frenta a malaria y proteómica redox en "Plasmodium falciparum"

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, leída el 29-04-2008.Fac. de Ciencias BiológicasTRUEProQuestpu

Novel insights into telomere biology and virulence gene expression in plasmodium falciparum

Plasmodium falciparum malaria is still one of the most preeminent and deadliest infectious diseases worldwide, imposing a tremendous health and economic burden on endemic countries. The high virulence of P. falciparum is mostly attributable to the expression of P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of infected red blood cells. PfEMP1 mediates intravascular parasite sequestration in vital organs, which contributes substantially to severe disease and death. Mutually exclusive transcription of the 60 var genes (encoding PfEMP1) and switching to formerly silenced variants results in antigenic variation and allows the parasite to efficiently evade host immune responses and to establish chronic infection. Members of the var multigene family are predominantly positioned close to chromosome ends. Characteristically, these regions are transcriptionally inert and demarcated by the repressive histone mark H3K9me3 and the evolutionary conserved silencing factor P. falciparum heterochromatin protein 1 (PfHP1). It is believed that this specialised environment at chromosome ends generates a structural framework for the epigenetic control of var gene expression. Moreover, telomeres play a crucial role in preserving genome integrity by protecting chromosome ends from inappropriate fusion and recombination events, as well as in regulating telomere length. However, we still lack a detailed functional understanding of the underlying molecular mechanisms that regulate Plasmodium chromosome end biology. During my PhD thesis, I tackled chromosome end biology from three different angles to improve our understanding of how virulence gene expression is regulated and how genome integrity is preserved. In a first project I performed an in-depth functional analysis of the epigenetic silencing factor PfHP1 by generating an inducible loss-of-function mutant. We showed that upon PfHP1 depletion parasites display a complete breakdown of mutually exclusive var expression and antigenic variation. Intriguingly, we also found that over 50% of PfHP1-deprived parasites represented viable gametocytes that complete sexual development up to stage V maturity. This high conversion rate was linked to the targeted de-repression of the ap2-g locus that codes for the ApiAP2 transcription factor AP2-G, which is essential for gametocyte conversion. Thus, our data unveiled PfHP1 not only as a master regulator of variegated expression of exported virulence factors, but also as a crucial factor in the regulation of sexual cell differentiation. In a second project I aimed at the functional characterisation of the chromosome-end associated protein PfSIP2, which was shown to specifically interact with SPE2 elements in subtelomeric regions. In-depth analysis of the expression profile of endogenous PfSIP2 revealed that this protein is only expressed during a very narrow time window of approximately 10hrs in late stage parasites, which coincides with intra-erythrocytic schizogony. Genome-wide ChIP-Seq experiments confirmed the exclusive binding of endogenous PfSIP2 to subtelomeric SPE2 landmarks in upsB var promoter regions and subtelomeric non-coding regions. Surprisingly, however, neither phenotypic changes nor differential gene expression were observed in a conditional PfSIP2-loss-of-function mutant and hence this approach didn’t uncover novel insights into the function of this ApiAP2 factor. In a third project I aimed at the identification of the telomere repeat-binding factor (TRF) in P. falciparum. Although TRFs are highly conserved and play essential roles in preserving chromosome integrity and regulating chromosome length in model eukaryotes, so far no TRF homologue has been found in the malaria parasites. My work reports about the successful de novo identification of the P. falciparum telomere repeat-binding protein (PfTRF). Intriguingly, this protein appears to be evolutionary distinct from TRFs in other eukaryotes as it binds to telomere repeat DNA via a C-terminal C2H2-type zinc finger domain instead of a MYB domain. Genome-wide mapping by ChIP-Seq experiments not only confirmed that PfTRF indeed binds to all chromosome termini in vivo, but as well revealed an unexpected second binding hotspot at telomere repeat-like sequences found in subtelomeric var gene promoters. A comprehensive characterisation of PfTRF using a conditional loss-of-function mutant identified essential roles for this protein in mitotic cell cycle progression and telomere length regulation. Hence, our findings provide important new insight into mechanisms underlying genome maintenance and possibly virulence gene silencing in P. falciparum. They further suggest that malaria parasites employ an evolutionary divergent molecular complex to preserve telomere function. In summary, my results provide important new and detailed understanding of the molecular processes involved in genome maintenance, virulence gene expression and sexual conversion in P. falciparum, processes that are highly relevant for malaria pathogenesis, parasite viability and malaria transmission. I am confident that these findings have important implications for the development of intervention strategies targeting parasite propagation and transmission

Dianas terapéuticas frenta a malaria y proteómica redox en "Plasmodium falciparum"

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, leída el 29-04-2008.Fac. de Ciencias BiológicasTRUEProQuestpu

Aspects of gene expression and regulation in plasmodium falciparum gametocytogenesis

Student Number : 9503239E - PhD thesis - Faculty of ScienceMalaria is one of the most debilitating pathogenic infections known to man, responsible for approximately three million deaths annually, primarily children in sub-Saharan Africa. The parasite has evaded multiple attempts at eradication, predominantly through the complexity of its life cycle, the ability to elude host immune response, and gametocyte formation to ensure dissemination. The recent completion of the genome sequence has opened up a multitude of avenues for exploration and identification of novel drug and vaccine targets, as well as providing a glimpse into the complex mechanisms that have contributed to the success of this pathogen. The mechanisms of gene regulation, especially those governing gametocytogenesis, have, however, not yet been elucidated. In this research, differential display has been used to identify some of the genes that are differentially expressed between the asexual parasite and gametocyte stages of P. falciparum. Numerous genes involved in diverse aspects of metabolism, protein synthesis and immune evasion were identified. A combination of BLASTN and BLASTX similarity searches was used to categorize and increase the confidence with which a transcript could be identified. Expression data for confidently identified genes were confirmed using reverse slot blot and available microarray data. PfMyb2, a novel transcription factor which may regulate genes involved in gametocytogenesis, was characterized. The DNA binding domains of the protein were cloned and expressed as a histidine fusion protein. Mobility shift assays were used to assess the in vitro binding capability of the recombinant 6xHis-PfMyb2, which bound to oligonucleotides containing the consensus Myb regulatory element. Two of the oligonucleotides represent sequences located within promoters of P. falciparum genes (Pfcrk1 and Pfmap1) known to play a role in regulating the cell cycle, a function ascribed to many members of the vertebrate Myb family. The identification of PfMyb2 as a bona fide transcription factor is a first step into gaining some insight into the many regulatory processes that occur during the life cycle of this complex organism. A better understanding of the molecular mechanisms that govern its survival is essential for the ultimate eradication of this deadly parasite

Novel malaria parasite proteins involved in erythrocyte invasion

Erythrocyte invasion is a key step in the Plasmodium life cycle. This process is tightly regulated, involving the sequential release of specialised apical secretory organelles – the micronemes, rhoptries and dense granules. These organelles contain proteins required for invasion and establishment of the parasitophorous vacuole, but most of the proteins remain uncharacterised. The aim of this project was to uncover novel proteins with a role in invasion by the human malaria parasite Plasmodium falciparum merozoites. I identified proteins using the following selection criteria: a) expression in the schizont/merozoite form of the parasite; b) conservation across the genus; c) the presence of a signal peptide and d) one or more transmembrane (TM) domains. A list of 64 proteins was identified, and filtered further based on novelty, presence in the merozoite proteome, expression in other life cycle stages, and difficulty of study. Five proteins were selected, and I produced recombinant protein and raised antibodies against three, which I used to identify the sub-cellular location of the protein within the parasite. The proteins appear to reside in either the rhoptries or the endoplasmic reticulum of the merozoite. Attempts were made to epitope-tag and delete all 3 genes, with a focus on one protein, the type IV Hsp40, PF11_0443. This protein contains two TM domains and is expressed during schizogony. By immunofluorescence it is present in the ER of early schizonts, before accumulating at the apex of merozoites in a rhoptry location. Immunoprecipitation experiments indicated that the protein binds known rhoptry proteins and other chaperones. The protein has been epitope-tagged but attempts to delete the gene by genetic recombination were unsuccessful. The gene is conserved in Plasmodium spp. and there are orthologues in higher eukaryotes, but it is absent from other Apicomplexa. Current studies are focused on the role of this protein in erythrocyte invasion

Etude différentielle des protéines membranaires exprimées à la surface de l'hématie infectée par Plasmodium falciparum, en fonction de la symptomatologie

La virulence de Plasmodium falciparum est fonction de sa capacité à séquestrer les érythrocytes infectés (iEs) dans les organes profonds de l hôte. Elle est liée à la mise en place d un trafic protéique conséquent au niveau membranaire et sub-membranaire de l iE. Cet adressage protéique aboutit à l expression d antigènes variant de surface, dont P. falciparum Erythrocyte Membrane Protein-1, PfEMP-1. Cet adhésineprésente une affinité pour divers récepteurs de l'hôte impliqués dans la physiopathologie des formes graves, telles que le paludisme associé à la grossesse (PAM) et le paludisme cérébral (CM). La diversité de liaison de PfEMP-1 est associée à la variabilité de séquence de son domaine extracellulaire. Ce domaine est constitué d une succession de domaines DBL et CIDR en nombre variable. PfEMP-1 est codée par les gènes var classifiés en groupes Ups A, B, C, E et B/A, B/C. Des régions de PfEMP-1 constituées d une succession établie de 2 à 4 domaines, nommées Domain Cassette, DC sont retrouvées dans différents isolats. L obtention de données exhaustives par LC-MS/MS a permis d établir un comparatif du protéome membranaire et hypothétique à partir des échantillons PAM et CM en comparaison à des accès simples de paludisme (UM). L identification protéique des PfEMP-1 a montré la singularité de l expression d une PfEMP-1 particulière, VAR2CSA chez les PAM. Ces résultats corroborent les analyses des transcrits du gène var2csa comme surexprimé chez les PAM, transcrit qu on retrouve également dans les infections de début et de fin de grossesse. Les PfEMP-1 identifiés au sein des CM étaient significativement associés aux groupes Ups A et B/A et la plupart des peptides identifiés étaient associés à la cassette DC8 dont le transcrit était également surexprimé. L analyse de filter-based feature selection a permis d identifier un sous ensemble de 13 et 14 protéines assignées comme protéines membranaires ou hypothétiques, prédictives des formes de paludisme PAM et CM, respectivement. Parmi ces protéines surexprimées chez les PAM, la présence de VAR2CSA valide l approche d analyse. PFI1785w a été identifiée et associée au PAM, quatre autres protéines apparaissent prédictives de cette forme clinique PFB0115w, PFF0325c, PFA_0410w et PF14_0018. Pour les CM, on distingue les protéines PfEMP-2 et antigen-332 comme spécifiques de cette forme clinique. Les autres protéines qui émergent de ce groupe sont des protéines de fonction inconnue, dont trois sont assignées comme des protéines exportées. L obtention et l analyse en LC-MS/MS d extraits protéiques issus d isolats de terrain font l originalité de ce travail et permettent la corrélation entre les données cliniques et biologiques. Cette étude confirme les données de transcrits sur la famille des gènes var et suggère de nouvelles intéractions protéiques dans le cadre du paludisme associé à la grossesse et cérébral.Plasmodium falciparum is responsible for severe malaria (cerebral malaria, CM and pregnancy associated malaria, PAM). During the intra-erythrocytic maturation of P. falciparum, parasite-derived proteins are expressed, exported and presented at the surface of the infected erythrocyte (iE) membrane. These include Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1). PfEMP-1 is a highly polymorphic adherence receptor, variants of which have been assigned to four groups (A-E) based on their sequence homology. Semi-conserved types, defined by tandem runs of specific domains ( domain cassettes (DC)), are also recognized. The PfEMP-1 type expressed determines the iE adherence phenotype, and is associated with the clinical outcome of infection. Parasite isolates from Beninese children or women presenting with, respectively, CM or PAM were collected along with samples from patients with uncomplicated malaria (UM). We assessed the transcript level of var genes by RT-qPCR and the expression of membrane and hypothetical proteins of Plasmodium by LC-MS/MS. Obtaining LC-MS/MS data enabled a comparison of hypothetical and membrane proteome samples from PAM and CM for comparison with UM samples. The proteomics-based identification of PfEMP-1 showed the expression of a particular PfEMP-1, VAR2CSA, in PAM isolates. These results corroborate the analysis of gene transcripts showing that var2csa is overexpressed in PAM, with transcripts found in isolates from infections both early and late in pregnancy. The PfEMP-1 variants identified in CM samples were predominantly from the Ups groups A and B/A, and most of the peptides identified by LC-MS/MS were associated with the DC8 cassette for which the transcripts were also overexpressed. Analysis using filter-based feature selection identified subsets of 13 and 14 proteins, assigned either as hypothetical or membrane proteins that were predictive of PAM and CM syndromes respectively. The presence of VAR2CSA amongst the proteins overexpressed in PAM samples validates the analytical approach. PFI1785w has previously been identified and associated with the PAM, whilst four other proteins, PFB0115w, PFF0325c, PFA_0410w and PF14_0018, appear to be also predictive of the syndrome. PfEMP-2 protein and antigen-332 were found to be specifically expressed in CM samples. Three other proteins, assigned as exported but with unknown function, were also associated with CM samples. Obtaining and analyzing LC-MS/MS-derived data from protein extracts of field isolates represents the originality of this work and it allowed the identification of correlations between clinical and biological data. This study confirms the transcriptional data relating to the var gene family and provides evidence of new protein interactions in the context both of malaria associated with pregnancy and of cerebral malaria.PARIS5-Bibliotheque electronique (751069902) / SudocPARIS-BIUM-Bib. électronique (751069903) / SudocSudocFranceF