Chromodomain Protein Interacts with H3K9me3 and Controls RBC Rosette Formation by Regulating the Expression of a Subset of RIFINs in the Malaria Parasite.

Plasmodium falciparum expresses clonally variant proteins on the surface of infected erythrocytes to evade the host immune system. The clonally variant multigene families include var, rifin, and stevor, which express Erythrocyte Membrane Protein 1 (EMP1), Repetitive Interspersed Families of polypeptides (RIFINs), and Sub-telomeric Variable Open Reading frame (STEVOR) proteins, respectively. The rifins are the largest multigene family and are essentially involved in the RBC rosetting, the hallmark of severe malaria. The molecular regulators that control the RIFINs expression in Plasmodium spp. have not been reported so far. This study reports a chromodomain-containing protein (PfCDP) that binds to H3K9me3 modification on P. falciparum chromatin. Conditional deletion of the chromodomain (CD) gene in P. falciparum using an inducible DiCre-LoxP system leads to selective up-regulation of a subset of virulence genes, including rifins, a few var, and stevor genes. Further, we show that PfCDP conditional knockout (PfΔCDP) promotes RBC rosette formation. This study provides the first evidence of an epigenetic regulator mediated control on a subset of RIFINs expression and RBC rosetting by P. falciparum.

A 39-Amino-Acid C-Terminal Truncation of GDV1 Disrupts Sexual Commitment in Plasmodium falciparum.

Malaria is a mosquito-borne disease caused by apicomplexan parasites of the genus Plasmodium. Completion of the parasite's life cycle depends on the transmission of sexual stages, the gametocytes, from an infected human host to the mosquito vector. Sexual commitment occurs in only a small fraction of asexual blood-stage parasites and is initiated by external cues. The gametocyte development protein 1 (GDV1) has been described as a key facilitator to trigger sexual commitment. GDV1 interacts with the silencing factor heterochromatin protein 1 (HP1), leading to its dissociation from heterochromatic DNA at the genomic locus encoding AP2-G, the master transcription factor of gametocytogenesis. How this process is regulated is not known. In this study, we have addressed the role of protein kinases implicated in gametocyte development. From a pool of available protein kinase knockout (KO) lines, we identified two kinase knockout lines which fail to produce gametocytes. However, independent genetic verification revealed that both kinases are not required for gametocytogenesis but that both lines harbor the same mutation that leads to a truncation in the extreme C terminus of GDV1. Introduction of the identified nonsense mutation into the genome of wild-type parasite lines replicates the observed phenotype. Using a GDV1 overexpression line, we show that the truncation in the GDV1 C terminus does not interfere with the nuclear import of GDV1 or its interaction with HP1 in vitro but appears to be important to sustain GDV1 protein levels and thereby sexual commitment.IMPORTANCE Transmission of malaria-causing Plasmodium species by mosquitos requires the parasite to change from a continuously growing asexual parasite form growing in the blood to a sexually differentiated form, the gametocyte. Only a small subset of asexual parasites differentiates into gametocytes that are taken up by the mosquito. Transmission represents a bottleneck in the life cycle of the parasite, so a molecular understanding of the events that lead to stage conversion may identify novel intervention points. Here, we screened a subset of kinases we hypothesized to play a role in this process. While we did not identify kinases required for sexual conversion, we identified a mutation in the C terminus of the gametocyte development 1 protein (GDV1), which abrogates sexual development. The mutation destabilizes the protein but not its interaction with its cognate binding partner HP1. This suggests an important role for the GDV1 C terminus beyond trafficking and protein stability.

An exported kinase family mediates species-specific erythrocyte remodelling and virulence in human malaria.

The most severe form of human malaria is caused by Plasmodium falciparum. Its virulence is closely linked to the increase in rigidity of infected erythrocytes and their adhesion to endothelial receptors, obstructing blood flow to vital organs. Unlike other human-infecting Plasmodium species, P. falciparum exports a family of 18 FIKK serine/threonine kinases into the host cell, suggesting that phosphorylation may modulate erythrocyte modifications. We reveal substantial species-specific phosphorylation of erythrocyte proteins by P. falciparum but not by Plasmodium knowlesi, which does not export FIKK kinases. By conditionally deleting all FIKK kinases combined with large-scale quantitative phosphoproteomics we identified unique phosphorylation fingerprints for each kinase, including phosphosites on parasite virulence factors and host erythrocyte proteins. Despite their non-overlapping target sites, a network analysis revealed that some FIKKs may act in the same pathways. Only the deletion of the non-exported kinase FIKK8 resulted in reduced parasite growth, suggesting the exported FIKKs may instead support functions important for survival in the host. We show that one kinase, FIKK4.1, mediates both rigidification of the erythrocyte cytoskeleton and trafficking of the adhesin and key virulence factor PfEMP1 to the host cell surface. This establishes the FIKK family as important drivers of parasite evolution and malaria pathology.

A Novel Tool for the Generation of Conditional Knockouts To Study Gene Function across the Plasmodium falciparum Life Cycle.

Plasmodium falciparum has a complex life cycle that involves interaction with multiple tissues inside the human and mosquito hosts. Identification of essential genes at all different stages of the P. falciparum life cycle is urgently required for clinical development of tools for malaria control and eradication. However, the study of P. falciparum is limited by the inability to genetically modify the parasite throughout its life cycle with the currently available genetic tools. Here, we describe the detailed characterization of a new marker-free P. falciparum parasite line that expresses rapamycin-inducible Cre recombinase across the full life cycle. Using this parasite line, we were able to conditionally delete the essential invasion ligand AMA1 in three different developmental stages for the first time. We further confirm efficient gene deletion by targeting the nonessential kinase FIKK7.1.IMPORTANCE One of the major limitations in studying P. falciparum is that so far only asexual stages are amenable to rapid conditional genetic modification. The most promising drug targets and vaccine candidates, however, have been refractory to genetic modification because they are essential during the blood stage or for transmission in the mosquito vector. This leaves a major gap in our understanding of parasite proteins in most life cycle stages and hinders genetic validation of drug and vaccine targets. Here, we describe a method that supports conditional gene deletion across the P. falciparum life cycle for the first time. We demonstrate its potential by deleting essential and nonessential genes at different parasite stages, which opens up completely new avenues for the study of malaria and drug development. It may also allow the realization of novel vaccination strategies using attenuated parasites.

Specific expression and export of the Plasmodium falciparum Gametocyte EXported Protein-5 marks the gametocyte ring stage.

BACKGROUND: Plasmodium falciparum sexual development plays a fundamental role in the transmission and spread of malaria. The ability to generate gametocytes can be lost during culture in vitro, often associated with the loss of a subtelomeric region of chromosome 9. Gametocytogenesis starts with erythrocyte invasion by a sexually committed merozoite, but the first available specific marker of sexual differentiation appears only from 24 h post invasion. METHODS: Specific antibodies and gene fusions were produced to study the timing of expression and the sub-cellular localization of the P. falciparum Gametocyte EXported Protein-5 (PfGEXP5), encoded in the subtelomeric region of chromosome 9. Expression patterns were examined in wild-type parasites and in parasite lines mutated in the Apetala2-G (AP2-G) transcription factor, governing a cascade of early sexual stage specific genes. RESULTS: PfGEXP5 is highly expressed in early sexual stages and it is actively exported to the infected erythrocyte cytoplasm from as early as 14 h post-invasion in haemozoin-free, ring stage-like parasites. The pattern of PfGEXP5 expression and export is similar in wild-type parasites and in independent AP2-G defective parasite lines unable to produce gametocytes. CONCLUSIONS: PfGEXP5 represents the earliest post-invasion sexual stage marker described to date. This provides a tool that can be used to identify sexually committed ring stage parasites in natural infections. This early gametocyte marker would enable the identification and mapping of malaria transmission reservoirs in human populations and the study of gametocyte sequestration dynamics in infected individuals. The fact that regulation of PfGEXP5 does not depend on the AP2-G master regulator of parasite sexual development suggests that, after sexual commitment, differentiation progresses through multiple checkpoints in the early phase of gametocytogenesis.

Erythrocyte remodeling by Plasmodium falciparum gametocytes in the human host interplay.

The spread of malaria critically relies on the presence of Plasmodium transmission stages - the gametocytes - circulating in the blood of an infected individual, which are taken up by Anopheles mosquitoes. A striking feature of Plasmodium falciparum gametocytes is their long development inside the erythrocytes while sequestered in the internal organs of the human host. Recent studies of the molecular and cellular remodeling of the host erythrocyte induced by P. falciparum during gametocyte maturation are shedding light on how these may affect the establishment and maintenance of sequestration of the immature transmission stages and the subsequent release and circulation of mature gametocytes in the peripheral bloodstream.

Early gametocytes of the malaria parasite Plasmodium falciparum specifically remodel the adhesive properties of infected erythrocyte surface.

In Plasmodium falciparum infections the parasite transmission stages, the gametocytes, mature in 10 days sequestered in internal organs. Recent studies suggest that cell mechanical properties rather than adhesive interactions play a role in sequestration during gametocyte maturation. It remains instead obscure how sequestration is established, and how the earliest sexual stages, morphologically similar to asexual trophozoites, modify the infected erythrocytes and their cytoadhesive properties at the onset of gametocytogenesis. Here, purified P. falciparum early gametocytes were used to ultrastructurally and biochemically analyse parasite-induced modifications on the red blood cell surface and to measure their functional consequences on adhesion to human endothelial cells. This work revealed that stage I gametocytes are able to deform the infected erythrocytes like asexual parasites, but do not modify its surface with adhesive 'knob' structures and associated proteins. Reduced levels of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins are exposed on the red blood cell surface by these parasites, and the expression of the var gene family, which encodes 50-60 variants of PfEMP1, is dramatically downregulated in the transition from asexual development to gametocytogenesis. Cytoadhesion assays show that such gene expression changes and host cell surface modifications functionally result in the inability of stage I gametocytes to bind the host ligands used by the asexual parasite to bind endothelial cells. In conclusion, these results identify specific differences in molecular and cellular mechanisms of host cell remodelling and in adhesive properties, leading to clearly distinct host parasite interplays in the establishment of sequestration of stage I gametocytes and of asexual trophozoites.

Characterization of PAR1 and FGFR1 expression in invasive breast carcinomas: Prognostic significance.

Breast cancer is the most common cause of cancer mortality among women worldwide. Among the several factors associated with breast cancer development, angiogenesis plays an essential role and has currently emerged as a potential diagnostic, prognostic and therapeutic target. Protease-activated receptor 1 (PAR1) and fibroblast growth factor receptor 1 (FGFR1) have important activities in tumor angiogenesis and progression. The aim of this study was to investigate the prognostic significance of these two receptors, hypothesising significant correlations between receptor expression in tumor angiogenesis and clinicopathological parameters customarily used in breast cancer prognosis and prediction. Formalin-fixed and paraffin-embedded samples of ductal invasive breast carcinomas were used to analyze the expression of PAR1 and FGFR1, in the tumor cells as well as in the tumor stroma, and further determine intratumoral microvessel density (iMVD) to quantify intratumoral angiogenesis. Correlations between PAR1 and FGFR1 expression in tumor cells and stroma, iMVD and several clinicopathological parameters and molecular markers used in breast cancer diagnosis have been addressed. The correlation between PAR1 and FGFR1 suggests an association of the two receptors with a more aggressive breast cancer phenotype and, consequently, a potential role during tumor progression. The results reported in the present study also emphasize the importance of microenvironmental factors in tumor progression, while precluding the positive association between iMVD and breast cancer aggressiveness.

A switch in infected erythrocyte deformability at the maturation and blood circulation of Plasmodium falciparum transmission stages.

Achievement of malaria elimination requires development of novel strategies interfering with parasite transmission, including targeting the parasite sexual stages (gametocytes). The formation of Plasmodium falciparum gametocytes in the human host takes several days during which immature gametocyte-infected erythrocytes (GIEs) sequester in host tissues. Only mature stage GIEs circulate in the peripheral blood, available to uptake by the Anopheles vector. Mechanisms underlying GIE sequestration and release in circulation are virtually unknown. We show here that mature GIEs are more deformable than immature stages using ektacytometry and microsphiltration methods, and that a switch in cellular deformability in the transition from immature to mature gametocytes is accompanied by the deassociation of parasite-derived STEVOR proteins from the infected erythrocyte membrane. We hypothesize that mechanical retention contributes to sequestration of immature GIEs and that regained deformability of mature gametocytes is associated with their release in the bloodstream and ability to circulate. These processes are proposed to play a key role in P falciparum gametocyte development in the host and to represent novel and unconventional targets for interfering with parasite transmission.

Differential adhesive properties of sequestered asexual and sexual stages of Plasmodium falciparum on human endothelial cells are tissue independent.

The protozoan parasite Plasmodium falciparum, responsible for the most severe form of malaria, is able to sequester from peripheral circulation during infection. The asexual stage parasites sequester by binding to endothelial cell receptors in the microvasculature of various organs. P. falciparum gametocytes, the developmental stages responsible for parasite transmission from humans to Anopheles mosquitoes, also spend the almost ten days necessary for their maturation sequestered away from the peripheral circulation before they are released in blood mainstream. In contrast to those of asexual parasites, the mechanisms and cellular interactions responsible for immature gametocyte sequestration are largely unexplored, and controversial evidence has been produced so far on this matter. Here we present a systematic comparison of cell binding properties of asexual stages and immature and mature gametocytes from the reference P. falciparum clone 3D7 and from a patient parasite isolate on a panel of human endothelial cells from different tissues. This analysis includes assays on human bone marrow derived endothelial cell lines (HBMEC), as this tissue has been proposed as a major site of gametocyte maturation. Our results clearly demonstrate that cell adhesion of asexual stage parasites is consistently more efficient than that, virtually undetectable of immature gametocytes, irrespectively of the endothelial cell lines used and of parasite genotypes. Importantly, immature gametocytes of both lines tested here do not show a higher binding efficiency compared to asexual stages on bone marrow derived endothelial cells, unlike previously reported in the only study on this issue. This indicates that gametocyte-host interactions in this tissue are unlikely to be mediated by the same adhesion processes to specific endothelial receptors as seen with asexual forms.