Pietro Alano

Protein Export Marks the Early Phase of Gametocytogenesis of the Human Malaria Parasite Plasmodium falciparum

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.

Plasmodium falciparum gametocytes: still many secrets of a hidden life

Sexual differentiation and parasite transmission are intimately linked in the life cycle of malaria parasites. The specialized cells providing this crucial link are the Plasmodium gametocytes. These are formed in the vertebrate host and are programmed to mature into gametes emerging from the erythrocytes in the midgut of a blood‐feeding mosquito. The ensuing fusion into a zygote establishes parasite infection in the insect vector. Although key mechanisms of gametogenesis and fertilization are becoming progressively clear, the fundamental biology of gametocyte formation still presents open questions, some of which are specific to the human malaria parasite Plasmodium falciparum. Developmental commitment to sexual differentiation, regulation of stage‐specific gene expression, the profound molecular and cellular changes accompanying gametocyte specialization, the requirement for tissue‐specific sequestration in P. falciparum gametocytogenesis are proposed here as areas for future investigation. The epidemiological relevance of parasite transmission from humans to mosquito in the spread of malaria and of Plasmodium drug resistance genes indicates that understanding molecular mechanisms of gametocyte formation is highly relevant to design strategies able to interfere with the transmission of this disease.

Plasmodium falciparum transmission stages accumulate in the human bone marrow

Sexual stages of the human malaria parasite Plasmodium falciparum use the hematopoietic system of the bone marrow as a developmental niche. Digging Deep for Malaria Parasites Malaria remains a major public health problem in developing countries. The pathogenesis of the most deadly of human malaria parasites, Plasmodium falciparum, is related to the ability of infected red blood cells to sequester in the microvasculature of deep tissues. Using an existing tissue collection from malaria autopsy cases, Joice et al. now reveal that P. falciparum transmission stages sequester in the hematopoietic system of the human bone marrow. This finding suggests that new mechanisms may be involved in the sequestration of these transmission stages and that the hematopoietic system may be a major site of formation, development, and maturation of malaria transmission stages. Transmission of Plasmodium falciparum malaria parasites requires formation and development of gametocytes, yet all but the most mature of these sexual parasite forms are absent from the blood circulation. We performed a systematic organ survey in pediatric cases of fatal malaria to characterize the spatial dynamics of gametocyte development in the human host. Histological studies revealed a niche in the extravascular space of the human bone marrow where gametocytes formed in erythroid precursor cells and underwent development before reentering the circulation. Accumulation of gametocytes in the hematopoietic system of human bone marrow did not rely on cytoadherence to the vasculature as does sequestration of asexual-stage parasites. This suggests a different mechanism for the sequestration of gametocytes that could potentially be exploited to block malaria transmission.

PfPK7, an atypical MEK‐related protein kinase, reflects the absence of classical three‐component MAPK pathways in the human malaria parasite Plasmodium falciparum

Two members of the mitogen‐activated protein kinase (MAPK) family have been previously characterized in Plasmodium falciparum, but in vitro attempts at identifying MAP kinase kinase (MAPKK) homologues have failed. Here we report the characterization of a novel plasmodial protein kinase, PfPK7, whose top scores in blastp analysis belong to the MAPKK3/6 subgroup of MAPKKs. However, homology to MAPKKs is restricted to regions of the C‐terminal lobe of the kinase domain, whereas the N‐terminal region is closer to fungal protein kinase A enzymes (PKA, members of the AGC group of protein kinases). Hence, PfPK7 is a ‘composite’ enzyme displaying regions of similarity to more than one protein kinase family, similar to a few other plasmodial protein kinases. PfPK7 is expressed in several developmental stages of the parasite, both in the mosquito vector and in the human host. Recombinant PfPK7 displayed kinase activity towards a variety of substrates, but was unable to phosphorylate the two P. falciparum MAPK homologues in vitro, and was insensitive to PKA and MEK inhibitors. Together with the absence of a typical MAPKK activation site in its T‐loop, this suggests that PfPK7 is not a MAPKK orthologue, despite the fact that this enzyme is the most ‘MAPKK‐like’ enzyme encoded in the P. falciparum genome. This is consistent with recent observations that the plasmodial MAPKs are not true orthologues of the ERK1/2, p38 or JNK MAPKs, and strengthens the evidence that classical three‐component module‐dependent MAPK signalling pathways do not operate in malaria parasites, a feature that has not been described in any other eukaryote.

The Culture and Preparation of Gametocytes of Plasmodium falciparum for Immunochemical, Molecular, and Mosquito Infectivity Studies

Since the development of methods for in vitro cultivation of the asexual blood stages of P.faZciparum (1) and the production of mature gametocytes from such cultures capable of infecting mosquitoes (2), it has been possible to conduct a wide range of studies on the sexual stages of P. filciparum. These include studies on the infectivity of the gametocytes of this parasite to mosquitoes (3) and the biology of the parasites in the vector (4) including studies on transmission blocking immunity using monoclonal antibodies against gamete surface antigens (5) and human sera following natural malarial infections (6). The developmental origin of sexual stages of P. fulcipurum has been studied in culture (7) and biochemical and immunochemical studies have been done to identify and characterize sexual stage specific proteins, especially those that are targets of transmission blocking antibodies (5). The genes encoding several sexual stage specific proteins have been identified and fully sequenced (8, 9, 10).

Identification and Initial Characterization of Three Novel Cyclin-related Proteins of the Human Malaria Parasite Plasmodium falciparum

The molecular mechanisms regulating cell proliferation and development during the life cycle of malaria parasites remain to be elucidated. The peculiarities of the cell cycle organization during Plasmodium falciparum schizogony suggest that the modalities of cell cycle control in this organism may differ from those in other eukaryotes. Indeed, existing data concerning Plasmodium cell cycle regulators such as cyclin-dependent kinases reveal structural and functional properties that are divergent from those of their homologues in other systems. The work presented here lies in the context of the exploitation of the recently available P. falciparum genome sequence toward the characterization of putative cell cycle regulators. We describe the in silico identification of three open reading frames encoding proteins with maximal homology to various members of the cyclin family and demonstrate that the corresponding polypeptides are expressed in the erythrocytic stages of the infection. We present evidence that these proteins possess cyclin activity by demonstrating either their association with histone H1 kinase activity in parasite extracts or their ability to activate PfPK5, a P. falciparum cyclin-dependent kinase homologue, in vitro. Furthermore, we show that RINGO, a protein with no sequence homology to cyclins but that is nevertheless a strong activator of mammalian CDK1/2, is also a strong activator of PfPK5 in vitro. This raises the possibility that “cryptic” cell cycle regulators may be found among the 50% of the open reading frames in the P. falciparum genome that display no homology to any known proteins.

Gene inactivation of Pf11-1 of Plasmodium falciparum by chromosome breakage and healing: identification of a gametocyte-specific protein with a potential role in gametogenesis.

We report the identification of the product of the Plasmodium falciparum Pf11‐1 gene and demonstrate that it is a gametocyte‐specific protein that has a potential role in the rupture of the host erythrocyte and emergence of the gametes (gametogenesis). The Pf11‐1 gene is a large locus (30 kb) whose sequence predicts a glutamic acid‐rich polypeptide. Our identification of the Pf11‐1 gene product as gametocyte specific was greatly facilitated by the isolation of a mutant parasite clone in which greater than 90% of the Pf11‐1 gene was deleted. Molecular analysis of the mutant locus suggests that the underlying genetic mechanism is chromosome breakage and subsequent healing by the addition of telomere repeats. PCR‐based analysis showed that similar DNA rearrangements occur commonly in small subpopulations of most laboratory strains, suggesting that the Pf11‐1 locus represents a fragile chromosome region. Northern blot analysis demonstrates that a large Pf11‐1 gene‐specific transcript (much greater than 10 kb) is present in gametocytes but not in asexual blood stage parasites. The Pf11‐1 protein was localized by electron microscopy to granules in the cytoplasm of gametocytes adjacent to the membrane of the parasitophorous vacuole. Following in vitro stimulation of gametogenesis, the Pf11‐1 protein was found in the membrane of lysed erythrocytes, suggesting a role for Pf11‐1 in erythrocyte rupture within the mosquito gut.

The Plasmodium falciparum schizont phosphoproteome reveals extensive phosphatidylinositol and cAMP-protein kinase A signaling

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.

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.

A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection

OBJECTIVES Plasmodium gametocytes, responsible for malaria parasite transmission from humans to mosquitoes, represent a crucial target for new antimalarial drugs to achieve malaria elimination/eradication. We developed a novel colorimetric screening method for anti-gametocyte compounds based on the parasite lactate dehydrogenase (pLDH) assay, already standardized for asexual stages, to measure gametocyte viability and drug susceptibility. METHODS Gametocytogenesis of 3D7 and NF54 Plasmodium falciparum strains was induced in vitro and asexual parasites were depleted with N-acetylglucosamine. Gametocytes were treated with dihydroartemisinin, epoxomicin, methylene blue, primaquine, puromycin or chloroquine in 96-well plates and the pLDH activity was evaluated using a modified Makler protocol. Mosquito infectivity was measured by the standard membrane feeding assay (SMFA). RESULTS A linear correlation was found between gametocytaemia determined by Giemsa staining and pLDH activity. A concentration-dependent reduction in pLDH activity was observed after 72 h of drug treatment, whereas an additional 72 h of incubation without drugs was required to obtain complete inhibition of gametocyte viability. SMFA on treated and control gametocytes confirmed that a reduction in pLDH activity translates into reduced oocyst development in the mosquito vector. CONCLUSIONS The gametocyte pLDH assay is fast, easy to perform, cheap and reproducible and is suitable for screening novel transmission-blocking compounds, which does not require parasite transgenic lines.