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Parasite development in the mosquito
The parasite journey in the mosquito begins with the ingestion of infected blood in the mosquito midgut lumen, as shown in Figure 6. Plasmodium female and male gametocytes mature into gametes. Each male gametocyte by a process called exflagellation, generates eight haploid motile gametes. Exfagellation occurs principally due to temperature decrease and pH changes as well as through exposure to xanthurenic acid in the insect midgut (Billker et al., 1998). Next, male and female gamete fusion results in the formation of a zygote, which differentiates into a motile invasive stage called ookinete. The ookinete penetrates the peritrophic matrix (PM) and the mosquito midgut epithelium, passing from the apical to the basal suface, where it transforms into a young oocyst. Ookinete laminin, the major component of the basal lamina, is essential to traverse the epithelium and for oocyst development. Of note, at this stage, the parasite has to deal with the mosquito immune response for the first time, which is responsible for the high parasite mortality observed during ookinete-oocyst transition. Indeed, this phase represents the first bottleneck in the Plasmodium life cycle.
Plasmodium Sporozoites
Sporozoites represent the transmission stage between the mosquito and the mammalian host. Upon release from the oocyst, thousands of sporozoites migrate through different mosquito cells and membranes to finally colonize the salivary glands, where they can be stored for several days. Additionally, in the mammalian host, the sporozoites migrate through the skin and blood circulation towards the liver (Prudêncio and Mota, 2007), where they replicate to produce thousands of infectious merozoites. To optimally move, the sporozoite (represented in figure 7), is an elongated polarized cell, 10-15 μm long and 1 μm in diameter. The cell is enveloped in a triple pellicle made by the plasma membrane and the underlying IMC, with the shape of the sporozoite maintained by microtubules. Sporozoites contain two sets of secretory organelles, the rhoptries and the micronemes. The rhoptries are large, paired, pear-shaped organelles filled with proteins and phospholipids. The micronemes are small vesicles with a neck-like extension. Microneme proteins are typically involved in gliding motility, adhesion to substrates and host cell recognition, while rhoptry proteins are implicated in entry into the host cell and parasitophorous vacuole formation (Baum et al., 2006, p. 200; Counihan et al., 2013; Santos et al., 2009).
Sinusoid Traversal
The parasite glides along the sinusoid, frequently moving against the bloodstream, until it recognizes select chondroitin and heparan sulfate proteoglycans on the surface of Kupffer cells (KCs) via CSP (Pradel et al., 2002). Frevert and collaborators proposed the gateway model hypothesizing that, sporozoites translocate across the sinusoidal barrier exclusively through KCs inside a non-fusogenic PV. In keeping with this, it has been observed that sporozoites can migrate through cells (Mota and Rodriguez, 2001), with genetic studies in P. berghei identifying several parasite factors involved in the process of cell traversal (CT). However, recent intravital imaging studies demonstrated that KCs do not constitute a mandatory gateway for Plasmodium sporozoites (Tavares et al., 2013). The authors showed that sporozoites can cross the liver sinusoidal barrier by multiple mechanisms, most of which are associated with cell traversal activity through endothelial cells (EC) and/or Kupffer cells. However, CT-deficient mutants still maintain a residual capacity to cross sinusoidal barriers (Ishino et al., 2005; Kariu et al., 2006), and the mechanisms used might correspond to either a paracellular pathway, as suggested for Toxoplasma tachyzoite transmigration (Barragan et al., 2005) or a transcellular pathway, through the formation of channels inside EC, like those generated by extravasating leukocytes (Carman and Springer, 2004) or through manipulating the fenestrations of liver sinusoidal EC (Warren et al., 2007). Finally, the sporozoite crosses the space of Disse to continue its path to the liver parenchyma where it encounters hepatocytes.
Invasion of Hepatocytes
The sporozoite transmigrates through several hepatocytes before homing in on its final niche hepatocyte. Here, it switches to productive invasion and differentiates into an exoerythrocytic form (EEF). A sequence of steps occurs during productive invasion: initially, a “distant” attachment is mediated by proteins secreted by micronemes. The rhoptry proteins are then secreted to form the moving junction (MJ), followed by engagement of the parasite actin-myosin motor, leading to parasite internalization. The parasite forms a parasitophorous vacuole (PV) (Besteiro et al., 2011), enclosed in a membrane (PVM), where its development takes place. Inside the vacuole the parasite is protected from the host immune response and can undergo schizogonic development. At this stage, the parasite nucleus divides multiple times with a concomitant syncytial organisation without cell segmentation. After 2 days or 6- 7 days respectively for rodent and primate parasites, the schizont is mature and full of merozoites.
Exo-erythrocytic Merozoite Formation and Release
Formation of individual liver stage merozoites begins when the parasite plasma membrane invaginates to demarcate individual merozoites with a single haploid nucleus and the necessary organelles (Figure 10). The PVM then breaks down, releases merozoites and PV contents into the host cell cytoplasm, in turn activating host cell mitochondrial disintegration and inhibiting host cell protein biosynthesis. The host cell now detaches.
Genome organization and gene expression in P. falciparum
Given its remarkable life cycle (as discussed in Section 2) and its capability to colonize numerous host and insect cells, the Plasmodium spp. parasite needs to respond quickly to diverse and hostile environments, and persist in the host until transmission is achieved. Studies to understand how the parasite adapts, survives and propagates under varied growth environments revealed that it differentially regulates gene expression during its life cycle (Bozdech et al., 2003; Le Roch et al., 2003) , at the transcriptomic and proteomic levels.
This regulation is orchestrated by several mechanisms, including epigenetic, transcriptional, post-transcriptional and translational regulation. Due to the absence of most specialized eukaryotic transcription factors in the Plasmodium genome (Gardner et al., 2002a), the epigenetic machinery has an important role in gene expression regulation. Indeed, to date, the only known Plasmodium spp. DNA-binding transcriptional regulators belong to the 27- member Apicomplexan Aptela 2 or ApiAP2 family (Painter et al., 2011). While some ApiAP2 proteins are expressed in a stage-specific manner, a subset of them are expressed throughout the four stages of the intraerythrocytic cycle (ring, trophozoite, early schizont and late schizont stage). The predicted DNA-binding AP2 domain is ∼60 aa in size and can be found alone, or in tandem with one or two additional AP2 domains. These domains bind with high specificity to unique DNA motifs, which are typically found in the upstream promoter regions of distinct sets of genes.
P. falciparum Chromosome Organization and Structure
The sequencing of the P. falciparum genome performed in 2002 revealed that the nuclear genome has 5,300 protein-encoding genes (Gardner et al., 2002a); the most recent annotation contains 5700 protein-coding genes and select groups of non-conding genes. In addition, it has two organellar genomes, the mitochondrial genome of 6 kb and the nonphotosynthetic plant derived apicoplast genome of 35 kb.
The highly AT-rich nuclear haploid genome of P. falciparum is 23.0 Mb is size and organized into 14 linear chromosomes (Gardner et al., 2002b). The chromosomes have a size ranging from 0.643 Mb for chromosome 1 to 3.29 Mb for chromosome 14. Each chromosome is compartmentalized, containing conserved regions in its central domain where the housekeeping genes are located, and polymorphic regions at its telomeric ends where highly variable gene families cluster (Scherf et al., 2008a). Chromosomes ends are made of tandem telomeric repeats (GGGTT(T/C)A), followed by an array of noncoding and coding DNA elements, the so-called telomere-associated sequences (TAS), in the subtelomeric regions (Figure 12). The terminal tract of P. falciparum telomeric DNA is assembled into a nonnucleosomal chromatin structure, the telosome (Figueiredo et al., 2000) that can contribute to epigenetic regulation. Moreover, the non-coding regions of TAS are composed of a mosaic of six different blocks of repetitive sequences that are located between the telomere and the coding region of TAS; these elements are called “telomere associated repetitive elements” (TAREs) and span 20-40 kb (Table 1) (Figueiredo et al., 2000; Scherf et al., 2001). TARE6, also known as rep20 based on the minimal repeat unit, is responsible for chromosome-end size polymorphism. Notably, adjacent to the non-coding TAREs are located member of multigene families such as var, rif and stevor.
Chromatin and its Regulation in P. falciparum
In Plasmodium spp., chromatin is organized as in other eukaryotic organisms, into fundamental units of ∼147 bp of DNA wrapped around an histone octamer; these units are called nucleosomes and serve as binding sites for transcription factors to regulate gene expression (Cary et al., 1994). The P. falciparum genome encodes four canonical histones required to assemble the nucleosome (H2A, H2B, H3 and H4) and four histone variants (H2A.Z, H2Bv, H3.3 and CenH3); however its lacks a gene encoding for histone H1, the linker histone (Miao et al., 2006). This partially explains the lack of higher order compaction of nuclear DNA in P. falciparum. Histones H3 and H4 contain N-terminal tails that are sites of post-translational modifications (PTMs), covalent alterations of specific amino acid residues that involve addition or removal of chemical groups mediated by specific enzymes, By massspectrometry, PTMs such as acetylation and methylation have been identified in Plasmodium Histone H3 and H4 at different positions (Figure 12) (Cui and Miao, 2010). These are summarized in Table 2.
Table of contents :
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
LIST OF FIGURES
LIST OF TABLES
SUMMARY
INTRODUCTION
1. Introduction to Malaria
1.1 Malaria Epidemiology
1.1.1 Malaria Disease
1.1.2 Chronicity of Malaria Infection
1.2 Current Situation and Challenges of Malaria Control
1.2.1 Drug Treatments
1.2.2 Vaccine Candidates in Development
1.2.2.1 Subunit Vaccines
1.2.2.2 Live Attenuated Parasite Vaccines
2. Etiologic Agent and Plasmodium Biology
2.1 Plasmodium Life Cycle
2.2 Parasite development in the mosquito
2.2.1 Plasmodium Sporozoites
2.3 Infection of the Mammalian Host
2.3.1 Skin Stage
2.3.2 Liver Stage
2.3.2.1 Arrest in the Sinusoid
2.3.2.2 Sinusoid Traversal
2.3.2.3 Invasion of Hepatocytes
2.3.3 Exo-erythrocytic Merozoite Formation and Release
2.3.4 Blood Stage
3. Genome organization and gene expression in P. falciparum
3.1 P. falciparum Chromosome Organization and Structure
3.2 Chromatin and its Regulation in P. falciparum
3.3 Transcription and Post-transcriptional Gene Regulation
4. Epigenetic Regulation in P. falciparum
4.1 Histone Modifications and their Writers and Eraser
4.1.1 Mechanisms of Histone Modifications
4.1.1.1 Acetylation
4.1.1.2 Deacetylation
4.1.1.3 Lysine methylation
4.1.1.4 Lysine Demethylation
4.1.2 P. falciparum Hetrochromatin Protien 1 (PfHP1) and other histone modifications readers
4.2 Nuclear Organization
4.3 Epigenetic Control of Sexual Commitment
4.4 Epigenetic Control of Hypnozoites
5. Biology and Regulation of P. falciparum Erythrocyte Membrain Protein 1 (PfEMP1)
5.1 PfEMP1 function
5.2 PfEMP1/var gene organization ans structure
5.3 Classification of adhesive domains in PfEMP1
5.3.1 Type 3 PfEMP1
5.3.2 DC4-Type PfEMP1
5.3.3 DC5-Type PfEMP1
5.3.4 DC8- and DC13-Type PfEMP1
5.3.5 var2CSA-Type PfEMP1
5.4 var gene Transcription and its Regulation
OBJECTIVES
RESULTS
ARTICLE 1 Plasmodium falciparum full life cycle and Plasmodium ovale liver stages in humanized mice.
ARTICLE 2 Plasmodium falciparum PfSET7: enzymatic characterization and cellular localization of a novel protein methyltransferase in sporozoite, liver and erythrocytic stage parasite.
ARTICLE 3 A SMYD-type methyltransferase PfSET6 associates with a subset of gene loci of
Plasmodium falciparum and forms multiple cytoplasmic foci during asexual, sexual blood stage and sporozoite stage.
ARTICLE 4 Analysis of the epigenetic landscape of Plasmodium falciparum reveals expression of a clonally variant PfEMP1 member on the surface of sporozoites.
DISCUSSION
1. Genome-wide ChIP-seq analysis of P. falciparum sporozoites
1.1 P. falciparum sporozoites epigenome
1.2 Clonally variant gene regulation
2 varsporo expression in P. falciparum sporozoites
2.1 Adhesive properties of sporozoites and PfEMP1
2.2 Immunogenic properties
3. PfEMP1 protein expression in P. falciparum liver stage
CONCLUSION & PERSPECTIVES
BIBLIOGRAPHY
