The molecular basis of sperm-oocyte membrane interactions during mammalian fertilization

Gamete membrane interactions begin with adhesion (binding) of the sperm to the oocyte plasma membrane and culminate with fusion of the membranes of the gametes, thus creating the zygote through the union of these two very different cells. This review summarizes the molecular and cell biology of the cell-cell interactions between mammalian gametes. Recent research studies have provided new insights into the complexity of these interactions and into the importance of multimeric molecular networks and optimal membrane order in both sperm and oocytes for successful fertilization. Molecules that will be highlighted include cysteine-rich secretory protein 1 (CRISP1) and ADAMs [fertilin alpha (ADAM1), fertilin beta (ADAM2) and cyritestin (ADAM3)] on sperm, and integrins, CD9, and other integrin-associated proteins on oocytes, as well as other molecules. The characteristics of these gamete molecules are summarized, followed by discussions of the experimental data that provide evidence for their participation in gamete membrane interactions, and also of the specific roles that these molecules might play. Insights from a variety of research areas, including gamete biology, cell adhesion, and membrane fusion, are put together for a tentative model of how sperm-oocyte adhesion and fusion occur. The clinical relevance of correct gamete membrane interactions is also noted.     

Baculovirus virions displaying Plasmodium berghei circumsporozoite protein protect mice against malaria sporozoite infection

The display of foreign proteins on the surface of baculovirus virions has provided a tool for the analysis of protein-protein interactions and for cell-specific targeting in gene transfer applications. To evaluate the baculovirus display system as a vaccine vehicle, we have generated a recombinant baculovirus (AcNPV-CSPsurf) that displays rodent malaria Plasmodium berghei circumsporozoite protein (PbCSP) on the virion surface as a fusion protein with the major baculovirus envelope glycoprotein gp64. The PbCSP-gp64 fusion protein was incorporated and oligomerized on the virion surface and led to a 12-fold increase in the binding activity of AcNPV-CSPsurf virions to HepG2 cells. Immunization with adjuvant-free AcNPV-CSPsurf virions induced high levels of antibodies and gamma interferon-secreting cells against PbCSP and protected 60% of mice against sporozoite challenge. These data demonstrate that AcNPV-CSPsurf displays sporozoite-like PbCSP on the virion surface and possesses dual potentials as a malaria vaccine candidate and a liver-directed gene delivery vehicle.     

Plasmodium vivax malaria: An unusual presentation

Acute renal failure, disseminated intravascular coagulation (DIC), acute respiratory distress syndrome (ARDS), hypoglycemia, coma, or epileptic seizures are manifestations of severe Plasmodium falciparum malaria. On the other hand, Plasmodium vivax malaria seldom results in pulmonary damage, and pulmonary complications are exceedingly rare. We report the case of a 42-year-old male living in a malaria-endemic area who presented with ARDS and was diagnosed as having Plasmodium vivax malaria. A diagnosis of Plasmodium vivax malaria was established by a positive Plasmodium LDH immunochromatographic assay while a negative PfHRP2 based assay ruled out P. falciparum malaria. After specific anti-plasmodial therapy and intensive supportive care, the patient recovered and was discharged from hospital. The use of NIPPV in vivax-malaria related ARDS was associated with a good outcome.     

Immunoglobulin A nephropathy associated with Plasmodium falciparum malaria

Glomerulonephritis occurs as a rare form of renal manifestation in Plasmodium falciparum malaria. Herein, we report a case of falciparum malaria-associated IgA nephropathy for the first time. A 49-yr old male who had been to East Africa was diagnosed with Plasmodium falciparum malaria. Microhematuria and proteinuria along with acute kidney injury developed during the course of the disease. Kidney biopsy showed mesangial proliferation and IgA deposits with tubulointerstitial inflammation. Laboratory tests after recovery from malaria showed disappearance of urinary abnormalities and normalization of kidney function. Our findings suggest that malaria infection might be associated with IgA nephropathy.     

The transcriptional program of synchronous gametogenesis in Chlamydomonas reinhardtii

Cells of Chlamydomonas reinhardtii undergo gametogenesis to produce sexually competent gametes under nitrogen-starved conditions. By using a synchronized system for gametogenesis of early G1 cells, several previously identified marker genes and 18 novel nitrogen-starved gametogenesis (NSG) genes isolated by macroarray analysis were placed into at least three temporal classes of expression. Early genes are induced transiently in the first 2 h after transfer to nitrogen-free medium. Middle genes are strongly induced between 3 h and 4 h after nitrogen removal, a time corresponding to the acquisition of mating competency, suggesting their involvement in the gamete program. Late genes are induced between 5 h and 8 h after nitrogen removal, a time after the completion of gametic differentiation, suggesting that they are not directly involved in the formation of sexually competent gametes. All of the 18 NSG genes examined are induced in both mating-type plus and minus gametes and about two-thirds of the genes are also expressed in the mitotic cell cycle, especially at S/M phases.     

Cerebral malaria caused by Plasmodium vivax in adult subjects

Cerebral malaria is a diffuse encephalopathy associated with seizures and status epilepticus which can occur in up to one-third of patients with severe malaria, particularly that caused by Plasmodium falciparum.In this article, we report three cases of Plasmodium vivax malaria (all adult male patients) complicated by seizures and symptoms of diffuse meningoencephalitis. Two patients had predominantly meningeal signs, while in the third patient the features were purely of encephalitis All cases were treated with artesunate. Usually, cerebral malaria is caused by P. falciparum, and rarely, cerebral malaria is a presenting complication or occurs during the course of P. vivax infection.     

Complement-mediated lysis of Plasmodium falciparum gametes by malaria-immune human sera is associated with antibodies to the gamete surface antigen Pfs230.

Antibodies to the sexual-stage surface antigens of Plasmodium falciparum, Pfs230 and Pfs48/45, can abolish the infectivity of gametes to mosquitoes; these antigens have been proposed as candidates for inclusion in a malaria transmission-blocking vaccine. One possible mechanism of antibody-mediated transmission blocking is complement-mediated gamete lysis. We have used a panel of human sera from geographically distinct regions where malaria is endemic to investigate whether this may be a mechanism of naturally acquired transmission-blocking immunity to P. falciparum. By immunoprecipitation, we have shown that antibody recognition of Pfs230 and Pfs48/45 is limited, despite universal exposure to P. falciparum gametocytes. In vitro complement-mediated lysis of P. falciparum gametes was positively associated with the presence of antibodies to Pfs230 but not with antibodies to the N-terminal region of the precursor molecule (Pfs260), which is shed from the gametocyte surface at the time of gametogenesis. Similarly, antibodies to two other gametocyte-specific proteins, Pfs48/45 and Pfg27/25, were not associated with gamete lysis. All sera which mediate gamete lysis contain immunoglobulin G1 (IgG1) and/or IgG3 antibodies to gamete surface proteins as determined by an enzyme-linked immunosorbent assay. These data suggest that Pfs230 is a major target of complement-fixing antibodies which may be important for antibody-mediated transmission-blocking immunity.     

A conserved domain targets exported PHISTb family proteins to the periphery of Plasmodium infected erythrocytes

During blood-stage infection, malaria parasites export numerous proteins to the host erythrocyte. The Poly-Helical Interspersed Sub-Telomeric (PHIST) proteins are an exported family that share a common ‘PRESAN’ domain, and include numerous members in Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi. In P. falciparum, PHIST proteins have been implicated in protein trafficking and intercellular communication. A number of PHIST proteins are essential for parasite survival. Here, we identify nine members of the PHISTb sub-class of PHIST proteins, including one protein known to be essential for parasite survival, that localise to the erythrocyte periphery. These proteins have solubility characteristics consistent with their association with the erythrocyte cytoskeleton. Together, an extended PRESAN domain, comprising the PRESAN domain and preceding sequence, form a novel targeting-domain that is sufficient to localise a protein to the erythrocyte periphery. We validate the role of this domain in RESA, thus identifying a cytoskeleton-binding domain in RESA that functions independently of its known spectrin-binding domain. Our data suggest that some PHISTb proteins may act as cross-linkers of the erythrocyte cytoskeleton. We also show for the first time that peripherally-localised PHISTb proteins are encoded in genomes of P. knowlesi and vivax indicating a conserved role for the extended PRESAN domain of these proteins in targeting to the erythrocyte periphery.     

Depletion of the Plasmodium berghei thrombospondin-related sporozoite protein reveals a role in host cell entry by sporozoites

The malaria parasite sporozoite stage develops in the mosquito vector and is transmitted to the mammalian host by bite. Sporozoites engage in multiple interactions with vector and host tissue on the journey from their oocyst origin to their final destination inside hepatocytes. Several malaria proteins have been identified that mediate sporozoite interactions with target tissues such as secreted and surface-associated ligands CSP and TRAP, which contain a thrombospondin type 1 repeat (TSR). Recently, we identified thrombospondin-related sporozoite protein (TRSP) in Plasmodium sporozoites, which exhibits a single TSR in its putative extracellular N-terminal region and is highly conserved among Plasmodium species. Here, we show using targeted gene disruption in the rodent malaria model Plasmodium berghei, that lack of TRSP has no effect on the asexual blood stage cycle, parasite transmission to the mosquito, sporozoite development and infection of mosquito salivary glands. However, analysis of TRSP knockout sporozoites in vitro and in vivo indicates that this protein has a significant role in hepatocyte entry and therefore liver infection. Thus, TRSP is an additional TSR-containing malaria parasite protein that is mainly involved in initial infection of the mammalian host.     

In vivo studies support the role of trafficking and cytoskeletal-binding motifs in the interaction of MESA with the membrane skeleton of Plasmodium falciparum-infected red blood cells

In red blood cells (RBCs) infected with the malaria parasite Plasmodium falciparum, a 19-residue region of the mature parasite-infected erythrocyte surface antigen (MESA) associates with RBC cytoskeleton protein 4.1R; an interaction essential for parasite survival. This region in MESA is adjacent to a host targeting motif found in other malaria parasite proteins exported to the membrane skeleton. To demonstrate function of these motifs in vivo, regions of MESA fused to a reporter were expressed in malaria parasites. Immunochemical analyses confirmed the requirement for both motifs in the trafficking and interaction of MESA with the cytoskeleton and demonstrates their function in vivo.     

A novel protein antigen of the malaria parasite Plasmodium falciparum, located on the surface of gametes and sporozoites

A Plasmodium falciparum cDNA clone was isolated of which the insert is transcribed at high rates as a 1.4-kb mRNA in the sexual stages of the malaria parasite. The cDNA clone contains a copy of a non-interrupted gene which codes for a protein of 157 amino acids (Mr = 16607). This 16-kDa protein does not contain repetitive sequences and is characterised by a putative N-terminal signal sequence, a hydrophobic membrane anchor sequence and a highly hydrophilic C-terminal region suggesting that it is an integral membrane protein. Rabbit antisera raised against a synthetic peptide covering amino acids 31-47 of the 16-kDa protein and against recombinant fusion proteins recognised the 16-kDa antigen in protein extracts of gametocytes, macrogamete/zygotes and sporozoites by Western blot analysis. The rabbit antisera also reacted with gametes, gametocytes and sporozoites in a standard immunofluorescence assay. By immunoelectron microscopy using the protein A-gold method the 16-kDa protein could be clearly visualised on the surface of macrogametes and sporozoites, whereas the antigen was not detectable in the asexual erythrocytic stages of the parasite. The 16-kDa antigen of P. falciparum therefore might have the potential to elicit a dual protective immune response against the sporozoite and sexual stage parasites.     

A case of imported Plasmodium ovale malaria

There have been reports in Korea of imported malaria cases of four Plasmodium species, but there has been no report of imported Plasmodium ovale malaria confirmed by molecular biological methods. We report an imported case of that was confirmed by Wright-Giemsa-stained peripheral blood smear and nested polymerase chain reaction targeting the small subunit ribosomal RNA gene. The amplified DNA was sequenced and compared with other registered P. ovale isolates. The isolate in this study was a member of the classic type group. The patient was a 44-yr-old male who had worked as a woodcutter in Côte d''Ivoire in tropical West Africa. He was treated with hydroxychloroquine and primaquine and discharged following improvement. In conclusion, P. ovale should be considered as an etiology in the imported malaria in Korea, because the number of travelers to P. ovale endemic regions has recently increased.     

Transmission patterns of chloroplast genes after polyethylene glycol-induced fusion of gametes in non-mating mutants of Chlamydomonas reinhardtii

Summary Polyethylene glycol-induced artificial fusions were made between gametes of opposite mating-type defective in sexual copulation capacity, and the inheritance patterns of chloroplast genes were analysed. Gametogenesis in non-agglutinating mutant strains was verified by a recently developed assay system (Saito et al. 1988) which distinguishes between the vegetative cell and the gamete. Although sexual crosses leading to the formation of vegetative zygotes (diploids) occurred very rarely in non-fusing mutant strain imp-1, gametes of non-agglutinating mutant strains, imp-2 and agl-1, did not fuse to form vegetative zygotes. The artificial fusions gave rise to diploid products which preferentially transmitted the chloroplast genes from the mating-type plus gamete. Non-motile cells grown on nitrogen-free agar plates — a cell type used as gametes by Matagne (1981) in artificial fusion experiments which exhibited patterns of chloroplast gene transmission very different from ours (Matsuda et al. 1983; this paper) — can be classified as vegetative, but not as gametic, as defined by the assay system. We demonstrate that even gametes, when placed on solid nitrogen-free medium, de-differentiate into vegetative cells and, after suspension in a liquid medium, differentiate once again into gametes.     

Two putative protein export regulators promote Plasmodium blood stage development in vivo

Protein export is considered an essential feature of malaria parasite blood stage development. Here, we examined five components of the candidate Plasmodium translocon of exported proteins (PTEX), a complex thought to mediate protein export across the parasitophorous vacuole membrane into the host cell. Using the murine malaria model parasite Plasmodium berghei, we succeeded in generating parasite lines lacking PTEX88 and thioredoxin 2 (TRX2). Repeated attempts to delete the remaining three translocon components failed, suggesting essential functions for EXP2, PTEX150, and heat shock protein 101 (HSP101) during blood stage development. To analyze blood infections of the null-mutants, we established a flow cytometry-assisted intravital competition assay using three novel high fluorescent lines (Bergreen, Beryellow, and Berred). Although blood stage development of parasites lacking TRX2 was affected, the deficit was much more striking in PTEX88 null-mutants. The multiplication rate of PTEX88-deficient parasites was strongly reduced resulting in out-competition by wild-type parasites. Endogenous tagging revealed that TRX2::tag resides in distinct punctate organelles of unknown identity. PTEX88::tag shows a diffuse intraparasitic pattern in blood stage parasites. In trophozoites, PTEX88::tag also localized to previously unrecognized extensions reaching from the parasite surface into the erythrocyte cytoplasm. Together, our results indicate auxiliary roles for TRX2 and PTEX88 and central roles for EXP2, PTEX150, and HSP101 during P. berghei blood infection.     

Secretory Mechanisms and Ca<Superscript>2+</Superscript> Signaling in Gametes: Similarities to Regulated Neuroendocrine Secretion in Somatic Cells and Involvement in Emerging Pathologies

Recent studies demonstrate that regulated secretion in probably all mammalian cells, from gonadotropes to gametes, utilizes similar signaling systems, intracellular Ca²⁺ regulation, Ca²⁺-dependent proteins, cytoskeletal participation, and SNARE-mediated fusion. Thus, highly specialized cells, like sperm and eggs, should no longer be considered to have evolved a cell-type specific secretory mechanism. In gametes, Ca²⁺-dependent proteins and enzymes transduce elevations of intracellular Ca²⁺ into secretory events, i.e., exocytosis of the acrosome in sperm and cortical granules in the egg. Just as secretory deficiencies have clinical consequences in endocrine and exocrine cells, failure of secretion of cortical granules or the acrosome can result in failure of normal fertilization or fertilization followed by abnormal development. With the advent of human in vitro fertilization, such gamete pathologies have been recently identified and have led to new clinical procedures to achieve normal fertilization and pregnancies. A better understanding of the common Ca²⁺-dependent secretory pathways in both gametes and somatic cells should be beneficial to investigating mis-regulation in either cell type.     

Identification of novel parasitophorous vacuole proteins in P. falciparum parasites using BioID

Malaria blood stage parasites develop within red blood cells where they are contained in a vacuolar compartment known as the parasitophorous vacuole (PV). This compartment holds a key role in the interaction of the parasite with its host cell. However, the proteome of this compartment has so far not been comprehensively analysed. Here we used BioID in asexual blood stages of the most virulent human malaria parasite Plasmodium falciparum to identify new proteins of the PV. The resulting proteome contained many of the already known PV proteins and validation by GFP-knock-in of 10 previously in P. falciparum uncharacterised hits revealed 5 new PV proteins and two with a partial PV localisation. This included proteins peripherally attached to the inner face of the PV membrane as well as proteins anchored in the parasite plasma membrane that protrude into the PV. Using selectable targeted gene disruption we generated mutants for 2 of the 10 candidates. In contrast we could not select parasites with disruptions for another 3 candidates, strongly suggesting that they are important for parasite growth. Interestingly, one of these included the orthologue of UIS2, a protein previously proposed to regulate protein translation in the parasite cytoplasm but here shown to be an essential PV protein. This work extends the number of known PV proteins and provides a starting point for further functional analyses of this compartment.