Monocytes contribute to the pro-inflammatory immune response during the blood stage of a Plasmodium falciparum infection, but their precise role in malaria pathology is not clear. Besides ...phagocytosis, monocytes are activated by products from P. falciparum infected erythrocytes (IE) and one of the activation pathways is potentially the NLR family pyrin domain containing 3 (NLRP3) inflammasome, a multi-protein complex that leads to the production of interleukin (IL)-1β. In cerebral malaria cases, monocytes accumulate at IE sequestration sites in the brain microvascular and the locally produced IL-1β, or other secreted molecules, could contribute to leakage of the blood-brain barrier. To study the activation of monocytes by IE within the brain microvasculature in an in vitro model, we co-cultured IT4var14 IE and the monocyte cell line THP-1 for 24 hours and determined whether generated soluble molecules affect barrier function of human brain microvascular endothelial cells, measured by real time trans-endothelial electrical resistance. The medium produced after co-culture did not affect endothelial barrier function and similarly no effect was measured after inducing oxidative stress by adding xanthine oxidase to the co-culture. While IL-1β does decrease barrier function, barely any IL-1β was produced in the co- cultures, indicative of a lack of or incomplete THP-1 activation by IE in this co-culture model.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Primaquine (PQ) is an essential antimalarial drug but despite being developed over 70 years ago, its mode of action is unclear. Here, we demonstrate that hydroxylated-PQ metabolites (OH-PQm) are ...responsible for efficacy against liver and sexual transmission stages of Plasmodium falciparum. The antimalarial activity of PQ against liver stages depends on host CYP2D6 status, whilst OH-PQm display direct, CYP2D6-independent, activity. PQ requires hepatic metabolism to exert activity against gametocyte stages. OH-PQm exert modest antimalarial efficacy against parasite gametocytes; however, potency is enhanced ca.1000 fold in the presence of cytochrome P450 NADPH:oxidoreductase (CPR) from the liver and bone marrow. Enhancement of OH-PQm efficacy is due to the direct reduction of quinoneimine metabolites by CPR with the concomitant and excessive generation of H
O
, leading to parasite killing. This detailed understanding of the mechanism paves the way to rationally re-designed 8-aminoquinolines with improved pharmacological profiles.
The asexual blood stages of Plasmodium falciparum cause the most lethal form of human malaria. During growth within an infected red blood cell, parasite multiplication and formation of invasive ...merozoites is called schizogony. Here, we present a detailed analysis of the phosphoproteome of P. falciparum schizonts revealing 2541 unique phosphorylation sites, including 871 novel sites. Prominent roles for cAMP-dependent protein kinase A- and phosphatidylinositol-signaling were identified following analysis by functional enrichment, phosphoprotein interaction network clustering and phospho-motif identification tools. We observed that most key enzymes in the inositol pathway are phosphorylated, which strongly suggests additional levels of regulation and crosstalk with other protein kinases that coregulate different biological processes. A distinct pattern of phosphorylation of proteins involved in merozoite egress and red blood cell invasion was noted. The analyses also revealed that cAMP-PKA signaling is implicated in a wide variety of processes including motility. We verified this finding experimentally using an in vitro kinase assay and identified three novel PKA substrates associated with the glideosome motor complex: myosin A, GAP45 and CDPK1. Therefore, in addition to an established role for CDPK1 in the motor complex, this study reveals the coinvolvement of PKA, further implicating cAMP as an important regulator of host cell invasion.
Despite over a century of study of malaria parasites, parts of the Plasmodium falciparum life cycle remain virtually unknown. One of these is the early gametocyte stage, a round shaped cell ...morphologically similar to an asexual trophozoite in which major cellular transformations ensure subsequent development of the elongated gametocyte. We developed a protocol to obtain for the first time highly purified preparations of early gametocytes using a transgenic line expressing a green fluorescent protein from the onset of gametocytogenesis. We determined the cellular proteome (1427 proteins) of this parasite stage by high accuracy tandem mass spectrometry and newly determined the proteomes of asexual trophozoites and mature gametocytes, identifying altogether 1090 previously undetected parasite proteins. Quantitative label-free comparative proteomics analysis determined enriched protein clusters for the three parasite developmental stages. Gene set enrichment analysis on the 251 proteins enriched in the early gametocyte proteome revealed that proteins putatively exported and involved in erythrocyte remodeling are the most overrepresented protein set in these stages. One-tenth of the early gametocyte-enriched proteome is constituted of putatively exported proteins, here named PfGEXPs (P. falciparum gametocyte-exported proteins). N-terminal processing and N-acetylation at a conserved leucine residue within the Plasmodium export element pentamotif were detected by mass spectrometry for three such proteins in the early but not in the mature gametocyte sample, further supporting a specific role in protein export in early gametocytogenesis. Previous reports and results of our experiments confirm that the three proteins are indeed exported in the erythrocyte cytoplasm. This work indicates that protein export profoundly marks early sexual differentiation in P. falciparum, probably contributing to host cell remodeling in this phase of the life cycle, and that gametocyte-enriched molecules are recruited to modulate this process in gametocytogenesis.
A hallmark of cerebral malaria (CM) is sequestration of Plasmodium falciparum-infected erythrocytes (IE) within the brain microvasculature. Binding of IE to endothelium reduces microvascular flow ...and, combined with an inflammatory response, perturbs endothelial barrier function, resulting in breakdown of the blood-brain barrier (BBB). Cytoadherence leads to activation of the endothelium and alters a range of cell processes affecting signaling pathways, receptor expression, coagulation, and disruption of BBB integrity. Here, we investigated whether CM-derived parasites elicit differential effects on human brain microvascular endothelial cells (HBMECs), as compared to uncomplicated malaria (UM)-derived parasites. Patient-derived IE from UM and CM clinical cases, as well as non-binding skeleton-binding protein 1 knockout parasites, were overlaid onto tumour necrosis factor (TNF)-activated HBMECs. Gene expression analysis of endothelial responses was performed using probe-based assays of a panel of genes involved in inflammation, apoptosis, endothelial barrier function, and prostacyclin synthesis pathway. We observed a significant effect on endothelial transcriptional responses in the presence of IE, yet there was no significant correlation between HBMEC responses and type of clinical syndrome (UM or CM). Furthermore, there was no correlation between HBMEC gene expression and both binding itself and level of IE binding to HBMECs, as we detected the same change in endothelial responses when employing both binding and non-binding parasites. Our results suggest that interaction of IE with endothelial cells in this co-culture model induces some endothelial responses that are independent of clinical origin and independent of the expression of the major variant antigen Plasmodium falciparum erythrocyte membrane protein 1 on the IE surface.IMPORTANCECerebral malaria (CM) is the most prevalent and deadly complication of severe Plasmodium falciparum infection. A hallmark of this disease is sequestration of P. falciparum-infected erythrocytes (IE) in brain microvasculature that ultimately results in breakdown of the blood-brain barrier. Here, we compared the effect of P. falciparum parasites derived from uncomplicated malaria (UM) and CM cases on the relative gene expression of human brain microvascular endothelial cells (HBMECs) for a panel of genes. We observed a significant effect on the endothelial transcriptional response in the presence of IE, yet there is no significant correlation between HBMEC responses and the type of clinical syndrome (UM or CM). Furthermore, there was no correlation between HBMEC gene expression and both binding itself and the level of IE binding to HBMECs. Our results suggest that interaction of IE with endothelial cells induces endothelial responses that are independent of clinical origin and not entirely driven by surface Plasmodium falciparum erythrocyte membrane protein 1 expression.
Osmiophilic bodies are membrane-bound vesicles, found predominantly in Plasmodium female gametocytes, that become progressively more abundant as the gametocyte reaches full maturity. These vesicles ...lie beneath the subpellicular membrane of the gametocyte, and the release of their contents into the parasitophorous vacuole has been postulated to aid in the escape of gametocytes from the erythrocyte after ingestion by the mosquito. Currently, the only protein known to be associated with osmiophilic bodies in Plasmodium falciparum is Pfg377, a gametocyte-specific protein expressed at the onset of osmiophilic body development. Here we show by targeted gene disruption that Pfg377 plays a fundamental role in the formation of these organelles, and that female gametocytes lacking the full complement of osmiophilic bodies are significantly less efficient both in vitro and in vivo in their emergence from the erythrocytes upon induction of gametogenesis, a process whose timing is critical for fertilization with the short-lived male gamete. This reduced efficiency of emergence explains the significant defect in oocyst formation in mosquitoes fed blood meals containing Pfg377-negative gametocytes, resulting in an almost complete blockade of infection.
Gametocytes of the protozoan Plasmodium falciparum ensure malaria parasite transmission from humans to the insect vectors. In their development, they produce the abundant specific protein Pfg27, the ...function and in vivo molecular interactions of which are unknown. Here we reveal a previously unreported localisation of Pfg27 in the gametocyte nucleus by immunoelectron microscopy and studies with HaloTag and Green Fluorescent Protein fusions, and identify a network of interactions established by the protein during gametocyte development. We report the ability of endogenous Pfg27 to form oligomeric complexes that are affected by phosphorylation of the protein, possibly through the identified phosphorylation sites, Ser32 and Thr208. We show that Pfg27 binds RNA molecules through specific residues and that the protein interacts with parasite RNA-binding proteins such as EF1α and PfH45. We propose a structural model for Pfg27 oligomerisation, based on the sequence and structural conservation here recognised between Pfg27 and sterile alpha motif. This study provides a molecular basis for Pfg27 to establish an interaction network with RNA and RNA-binding proteins and to govern its dynamic oligomerisation in developing gametocytes.
In the human malaria parasite Plasmodium falciparum, gametocyte maturation is a process remarkably longer than in other malaria species, accompanied by expression of 2-300 sexual stage-specific ...proteins. Disruption of several of their encoding genes so far showed that only the abundant protein Pfg27, produced at the onset of sexual differentiation, is essential for gametocyte production. In contrast with what has been previously described, here we show that P. falciparum pfg27 disruptant lines are able to undergo all stages of gametocyte maturation, and are able to mature into gametes. A fraction of Pfg27-defective gametocytes show, however, distinct abnormalities in intra- and extra-cellular membranous compartments, such as accumulation of parasitophorous vacuole-derived vesicles in the erythrocyte cytoplasm, large intracellular vacuoles and discontinuities in their trilaminar cell membrane. This work revises current knowledge on the role of Pfg27, indicating that the protein is not required for parasite entry into sexual differentiation, and suggesting that it is instead involved in maintaining cell integrity in the uniquely long gametocytogenesis of P. falciparum.
In skeletal muscle differentiation, the retinoblastoma protein (pRb) is absolutely necessary to establish definitive mitotic arrest. It is widely assumed that pRb is equally essential to sustain the ...postmitotic state, but this contention has never been tested. Here, we show that terminal proliferation arrest is maintained in skeletal muscle cells by a pRb-independent mechanism. Acute Rb excision from conditional knockout myotubes caused reexpression of E2F transcriptional activity, cyclin-E and -A kinase activities, PCNA, DNA ligase I, RPA, and MCM2, but did not induce DNA synthesis, showing that pRb is not indispensable to preserve the postmitotic state of these cells. Muscle-specific gene expression was significantly down-regulated, showing that pRb is constantly required for optimal implementation of the muscle differentiation program. Rb-deleted myotubes were efficiently reactivated by forced expression of cyclin D1 and Cdk4, indicating a functionally significant target other than pRb for these molecules. Finally, Rb removal induced no DNA synthesis even in pocket-protein null cells. Thus, the postmitotic state of myotubes is maintained by at least two mechanisms, one of which is pocket-protein independent.
Several chemokines have been shown to act as angiogenic molecules or to modulate the activity of growth factors such as fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor ...(VEGF). The detection of the CC chemokine receptor (CCR) 8 message in human umbilical vein endothelial cells (HUVECs) by reverse transcription– polymerase chain reaction (RT-PCR) and RNase protection assay (RPA), prompted us to investigate the potential role exerted by the CC chemokine I-309, a known ligand of such receptor, in both in vitro and in vivo angiogenesis assays. We show here that I-309 binds to endothelial cells, stimulates chemotaxis and invasion of these cells, and enhances HUVEC differentiation into capillary-like structures in an in vitro Matrigel assay. Furthermore, I-309 is an inducer of angiogenesis in vivo in both the rabbit cornea and the chick chorioallantoic membrane assay (CAM).
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