Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity

Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito -- early and late oocysts containing midgut sporozoites, and the mature, infectious salivary gland sporozoites. Despite the morphological similarity between midgut and salivary gland sporozoites, their proteomes are markedly different, in agreement with their increase in hepatocyte infectivity. The different sporozoite proteomes contain a large number of stage specific proteins whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may be essential for sporozoite infectivity to humans.     

Reconstructing the mosaic glycolytic pathway of the anaerobic eukaryote Monocercomonoides

All eukaryotes carry out glycolysis, interestingly, not all using the same enzymes. Anaerobic eukaryotes face the challenge of fewer molecules of ATP extracted per molecule of glucose due to their lack of a complete tricarboxylic acid cycle. This may have pressured anaerobic eukaryotes to acquire the more ATP-efficient alternative glycolytic enzymes, such as pyrophosphate-fructose 6-phosphate phosphotransferase and pyruvate orthophosphate dikinase, through lateral gene transfers from bacteria and other eukaryotes. Most studies of these enzymes in eukaryotes involve pathogenic anaerobes; Monocercomonoides, an oxymonad belonging to the eukaryotic supergroup Excavata, is a nonpathogenic anaerobe representing an evolutionarily and ecologically distinct sampling of an anaerobic glycolytic pathway. We sequenced cDNA encoding glycolytic enzymes from a previously established cDNA library of Monocercomonoides and analyzed the relationships of these enzymes to those from other organisms spanning the major groups of Eukaryota, Bacteria, and Archaea. We established that, firstly, Monocercomonoides possesses alternative versions of glycolytic enzymes: fructose-6-phosphate phosphotransferase, both pyruvate kinase and pyruvate orthophosphate dikinase, cofactor-independent phosphoglycerate mutase, and fructose-bisphosphate aldolase (class II, type B). Secondly, we found evidence for the monophyly of oxymonads, kinetoplastids, diplomonads, and parabasalids, the major representatives of the Excavata. We also found several prokaryote-to-eukaryote as well as eukaryote-to-eukaryote lateral gene transfers involving glycolytic enzymes from anaerobic eukaryotes, further suggesting that lateral gene transfer was an important factor in the evolution of this pathway for denizens of this environment.     

Cloning, expression and characterisation of a recombinant triosephosphate isomerase from Taenia solium

We isolated and characterised the cDNA that encodes the glycolytic enzyme, triosephosphate isomerase from Taenia solium. A 450 bp DNA fragment was obtained by the polymerase chain reaction using a cDNA from larval stage as template and degenerate oligonucleotides designed from conserved polypeptide sequences from TPIs of several organisms. The fragment was used to screen a T. solium larval stage cDNA library. The isolated cDNA, encoding a protein of 250 amino acids shares 44.8-59.6% positional identity with other known TPIs, in which the catalytic enzyme residues were conserved. The complete coding sequence of the T. solium TPI cDNA was cloned into the expression vector pRSET and expressed as a fusion protein with an N-terminal tail of six histidine residues. The catalytic activity of the purified protein was similar to other TPI enzymes. Northern and Southern blot analysis suggest that in T. solium, single gene exists for triosephosphate isomerase and that the gene is expressed in all stages of the parasite.     

Gene disruption of Plasmodium falciparum p52 results in attenuation of malaria liver stage development in cultured primary human hepatocytes

Difficulties with inducing sterile and long lasting protective immunity against malaria with subunit vaccines has renewed interest in vaccinations with attenuated Plasmodium parasites. Immunizations with sporozoites that are attenuated by radiation (RAS) can induce strong protective immunity both in humans and rodent models of malaria. Recently, in rodent parasites it has been shown that through the deletion of a single gene, sporozoites can also become attenuated in liver stage development and, importantly, immunization with these sporozoites results in immune responses identical to RAS. The promise of vaccination using these genetically attenuated sporozoites (GAS) depends on translating the results in rodent malaria models to human malaria. In this study, we perform the first essential step in this transition by disrupting, p52, in P. falciparum an ortholog of the rodent parasite gene, p36p, which we had previously shown can confer long lasting protective immunity in mice. These P. falciparum P52 deficient sporozoites demonstrate gliding motility, cell traversal and an invasion rate into primary human hepatocytes in vitro that is comparable to wild type sporozoites. However, inside the host hepatocyte development is arrested very soon after invasion. This study reveals, for the first time, that disrupting the equivalent gene in both P. falciparum and rodent malaria Plasmodium species generates parasites that become similarly arrested during liver stage development and these results pave the way for further development of GAS for human use.     

Effect of anaerobiosis and anhydrobiosis on the extent of glycolytic enzyme binding in Artermia embryos

The effect of anaerobiosis and anhydrobiosis on the extent of binding of glycolytic enzymes to the particulate fraction of the cell was studied in Artemia salina embryos. During control aerobic development, trehalase, phosphofructokinase and pyruvate kinase showed an increase in the percentage associated with the particulate fraction which is consistent with the carbohydrate-based metabolism of Artemia embryos. However, anaerobiosis resulted in decreased enzyme binding for six glycolytic enzymes; hexokinase, aldolase, pyruvate kinase and lactate dehydrogenase were the exceptions. Decreased enzyme binding was also observed after exposure to dehydrating conditions. The results suggest that glycolytic rate could be regulated by changes in the distribution of glycolytic enzymes between free and bound forms in Artemia embryos. This reversible interaction of glycolytic enzymes with structural proteins may account for part of the metabolic arrest observed during anaerobic dormancy and anhydrobiosis.Abbreviation pH_i intracellular concentration of H⁺ ions     

Imaging movement of malaria parasites during transmission by Anopheles mosquitoes

Malaria is contracted when Plasmodium sporozoites are inoculated into the vertebrate host during the blood meal of a mosquito. In infected mosquitoes, sporozoites are present in large numbers in the secretory cavities of the salivary glands at the most distal site of the salivary system. However, how sporozoites move through the salivary system of the mosquito, both in resting and feeding mosquitoes, is unknown. Here, we observed fluorescent Plasmodium berghei sporozoites within live Anopheles stephensi mosquitoes and their salivary glands and ducts. We show that sporozoites move in the mosquito by gliding, a type of motility associated with their capacity to invade host cells. Unlike in vitro, sporozoite gliding inside salivary cavities and ducts is modulated in speed and motion pattern. Imaging of sporozoite discharge through the proboscis of salivating mosquitoes indicates that sporozoites need to locomote from cavities into ducts to be ejected and that their progression inside ducts favours their early ejection. These observations suggest that sporozoite gliding allows not only for cell invasion but also for parasite locomotion in host tissues, and that it may control parasite transmission.     

Proteomics analysis and protein expression during sporozoite excystation of Cryptosporidium parvum (Coccidia, Apicomplexa)

Cryptosporidiosis, caused by coccidian parasites of the genus Cryptosporidium, is a major cause of human gastrointestinal infections and poses a significant health risk especially to immunocompromised patients. Despite intensive efforts for more than 20 years, there is currently no effective drug treatment against these protozoa. This study examined the zoonotic species Cryptosporidium parvum at two important stages of its life cycle: the non-excysted (transmissive) and excysted (infective) forms. To increase our understanding of the molecular basis of sporozoite excystation, LC-MS/MS coupling with a stable isotope N-terminal labeling strategy using iTRAQ reagents was used on soluble fractions of both non-excysted and excysted sporozoites, i.e. sporozoites both inside and outside oocysts were examined. Sporozoites are the infective stage that penetrates small intestinal enterocytes. Also to increase our knowledge of the C. parvum proteome, shotgun sequencing was performed on insoluble fractions from both non-excysted and excysted sporozoites. In total 303 C. parvum proteins were identified, 56 of which, hitherto described as being only hypothetical proteins, are expressed in both excysted and non-excysted sporozoites. Importantly we demonstrated that the expression of 26 proteins increases significantly during excystation. These excystation-induced proteins included ribosomal proteins, metabolic enzymes, and heat shock proteins. Interestingly three Apicomplexa-specific proteins and five Cryptosporidium-specific proteins augmented in excysted invasive sporozoites. These eight proteins represent promising targets for developing vaccines or chemotherapies that could block parasite entry into host cells.     

Enzyme variation in Eimeria species of the chicken.

A method for the biochemical identification of protozoa belonging to the genus Eimeria is described for the first time. Starch gel electrophoresis of the enzymes lactate dehydrogenase, glucose phosphate isomerase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase from parasite extracts revealed both intra- and inter-species differences when 11 strains representative of 6 species of Eimeria were examined. Oocysts were the most accessible parasite stage for investigation but sporozoites and merozoites of an embryo-adapted strain of E. tenella were also examined for enzyme activity.     

Studies on the regulation of enolases and compartmentation of cytosolic enzymes in Saccharomyces cerevisiae

Three enolase isoenzymes can be distinguished after electrophoresis of yeast crude extracts. After adding glucose to derepressed cells, there was a coordinated increase in the activity of enolase I and decrease in enolase II activity. Enolase I was found to be repressed and enolase II simultaneously induced by glucose. The third enolase activity remained unchanged and was identified as that of a hybrid enzyme. Enolase catalyses the first common step of glycolysis and gluconeogenesis. Gluconeogenic enolase I shows substrate inhibition for 2-phosphoglycerate (glycolytic substrate) and glycolytic enolase II is substrate-inhibited by phosphoenolpyruvate (gluconeogenic substrate). The gluconeogenic reaction was inhibited up to 45% by physiological concentrations of fructose 1,6-bisphosphate. To test for cytological compartmentation, a method was developed for isolating microsomes. Effective enrichment of rough and smooth endoplasmic reticulum was demonstrated by electron microscopy. No evidence was obtained for any compartmentation of either enolases or other glycolytic enzymes.     

Role of gregarine parasite Ascogregarina culicis (Apicomplexa: Lecudinidae) in the maintenance of Chikungunya virus in vector mosquito

Ascogregarina culicis and Ascogregarina taiwanensis are common gregarine parasites of Aedes aegypti and Aedes albopictus mosquitoes, respectively. These mosquito species are also known to transmit dengue and Chikungunya viruses. The sporozoites of these parasites invade the midgut epithelial cells and develop intracellularly and extracellularly in the gut to complete their life cycles. The midgut is also the primary site for virus replication in the vector mosquitoes. Therefore, studies were carried out with a view to determine the possible role of these gregarines in the vertical transmission of dengue and Chikungunya viruses from larval to adult stage. Experiments were performed by exposing first instar mosquito larvae to suspensions containing parasite oocysts and viruses. Since Ascogregarina sporozoites invade the midgut of first instar larvae, the vertical transmission was determined by feeding the uninfected first instar larvae on the freshly prepared homogenates from mosquitoes, which were dually infected with viruses and the parasite oocysts. Similarly, the role of protozoan parasites in the vertical transmission of viruses was determined by exposing fresh first instar larvae to the dried pellets of homogenates prepared from the mosquitoes dually infected with viruses and the parasite oocysts. Direct vertical transmission and the vertical transmission of CHIK virus through the oocyst of the parasites were observed in the case of Ae. aegypti mosquitoes. It is suggested that As. culicis may have an important role in the maintenance of CHIK virus during the inter-epidemic period.     

Differential Gene Expression and Protein Localization of Cryptosporidium parvum Fatty Acyl‐CoA Synthetase Isoforms

Cryptosporidium parvum is unable to synthesize fatty acids de novo, but possesses three long‐chain fatty acyl‐CoA synthetase (CpACS) isoforms for activating fatty acids. We have recently shown that these enzymes could be targeted to kill the parasite in vitro and in vivo. Here, we demonstrated that the CpACS genes were differentially expressed during the parasite life cycle, and their proteins were localized to different subcellular structures by immunofluorescence and immuno‐electron microscopies. Among them, CpACS1 displayed as an apical protein in sporozoites and merozoites, but no or little presence during the intracellular merogony until the release of merozoites, suggesting that CpACS1 probably functioned mainly during the parasite invasion and/or early stage of intracellular development. Both CpACS2 and CpACS3 proteins were present in all parasite life cycle stages, in which CpACS2 was present in the parasite and the parasitophorous vacuole membranes (PVM), whereas CpACS3 was mainly present in the parasite plasma membranes with little presence in the PVM. These observations suggest that CpACS2 and CpACS3 may participate in scavenging and transport of fatty acids across the PVM and the parasite cytoplasmic membranes, respectively.     

Effect of electrical stimulation post mortem of bovine muscle on the binding of glycolytic enzymes. Functional and structural implications.

The extent of binding of glycolytic enzymes to the particulate fraction of homogenates was measured in bovine psoas muscle before and after electrical stimulation. In association with an accelerated glycolytic rate on stimulation, there was a significant increase in the binding of certain glycolytic enzymes, the most notable of which were phosphofructokinase, aldolase, glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase. From the known association of glycolytic enzymes with the I-band of muscle it is proposed that electrical stimulation of anaerobic muscle increases enzyme binding to actin filaments. Calculations of the extent of enzyme binding suggest that significant amounts of enzyme protein, particularly aldolase and glyceraldehyde 3-phosphate dehydrogenase, are associated with the actin filaments. The results also imply that kinetic parameters derived from considerations of the enzyme activity in the soluble state may not have direct application to the situation in the muscle fibre, particularly during accelerated glycolysis.     

Electron microscopic studies on the interaction of rat Kupffer cells and Plasmodium berghei sporozoites

The interactions between Plasmodium berghei sporozoites and Kupffer cells in rat liver were studied by transmission electron microscopy. Between 10 and 45 min after inoculation, sporozoites were found in the process of entering Kupffer cells and inside phagolysosomes. The sporozoites entered the Kupffer cells by phagocytosis as determined by the presence of pseudopods and local accumulations of aggregated microfilaments and the resulting exclusion of other organelles in the phagocyte cytoplasm beneath the attached parasite. Sporozoites were taken up either with their anterior end first, or backwards. Scanning electron microscopy of in vitro sporozoite Kupffer cell interaction confirmed these observations. It was concluded that sporozoites are taken up in a normal phagocytic way by the Kupffer cells, regardless of their initial place of contact or position. Thirty min after inoculation sporozoites found in phagolysosomes were still morphologically intact but after 45 min we could encounter completely digested sporozoites.     

Plasmodium berghei sporozoites in nonreplicative vacuole are eliminated by a PI3P‐mediated autophagy‐independent pathway

The protozoan parasite Plasmodium, causative agent of malaria, invades hepatocytes by invaginating the host cell plasma membrane and forming a parasitophorous vacuole membrane (PVM). Surrounded by this PVM, the parasite undergoes extensive replication. Parasites inside a PVM provoke the Plasmodium‐associated autophagy‐related (PAAR) response. This is characterised by a long‐lasting association of the autophagy marker protein LC3 with the PVM, which is not preceded by phosphatidylinositol 3‐phosphate (PI3P)‐labelling. Prior to productive invasion, sporozoites transmigrate several cells and here we describe that a proportion of traversing sporozoites become trapped in a transient traversal vacuole, provoking a host cell response that clearly differs from the PAAR response. These trapped sporozoites provoke PI3P‐labelling of the surrounding vacuolar membrane immediately after cell entry, followed by transient LC3‐labelling and elimination of the parasite by lysosomal acidification. Our data suggest that this PI3P response is not only restricted to sporozoites trapped during transmigration but also affects invaded parasites residing in a compromised vacuole. Thus, host cells can employ a pathway distinct from the previously described PAAR response to efficiently recognise and eliminate Plasmodium parasites.     

Malarial proteases and host cell egress: an 'emerging' cascade

Malaria is a scourge of large swathes of the globe, stressing the need for a continuing effort to better understand the biology of its aetiological agent. Like all pathogens of the phylum Apicomplexa, the malaria parasite spends part of its life inside a host cell or cyst. It eventually needs to escape (egress) from this protective environment to progress through its life cycle. Egress of Plasmodium blood-stage merozoites, liver-stage merozoites and mosquito midgut sporozoites relies on protease activity, so the enzymes involved have potential as antimalarial drug targets. This review examines the role of parasite proteases in egress, in the light of current knowledge of the mechanics of the process. Proteases implicated in egress include the cytoskeleton-degrading malarial proteases falcipain-2 and plasmepsin II, plus a family of putative papain-like proteases called SERA. Recent revelations have shown that activation of the SERA proteases may be triggered by regulated secretion of a subtilisin-like serine protease called SUB1. These findings are discussed in the context of the potential for development of new chemotherapeutics targeting this stage in the parasite's life cycle.     

Highly dynamic host actin reorganization around developing Plasmodium inside hepatocytes

Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver.     

Surface-expressed enolases of Plasmodium and other pathogens

Enolase is the eighth enzyme in the glycolytic pathway, a reaction that generates ATP from phosphoenol pyruvate in cytosolic compartments. Enolase is essential, especially for organisms devoid of the Krebs cycle that depend solely on glycolysis for energy. Interestingly, enolase appears to serve a separate function in some organisms, in that it is also exported to the cell surface via a poorly understood mechanism. In these organisms, surface enolase assists in the invasion of their host cells by binding plasminogen, an abundant plasma protease precursor. Binding is mediated by the interaction between a lysine motif of enolase with Kringle domains of plasminogen. The bound plasminogen is then cleaved by specific proteases to generate active plasmin. Plasmin is a potent serine protease that is thought to function in the degradation of the extracellular matrix surrounding the targeted host cell, thereby facilitating pathogen invasion. Recent work revealed that the malaria parasite Plasmodium also expresses surface enolase, and that this feature may be essential for completion of its life cycle. The therapeutic potential of targeting surface enolases of pathogens is discussed.     

Enolase is present at the centrosome of HeLa cells.

Antibodies raised against the C-terminus and N-terminus region of gamma gamma enolase, as well as a polyclonal antibody raised against bovine brain gamma gamma enolase, were used to study the distribution of this glycolytic enzyme during the cell cycle in HeLa cells. Enolase was found to be present throughout the cytoplasm of both interphase and dividing cells. In addition, a portion of cellular enolase was detected at the centrosome throughout the cell cycle. The capacity of glycolytic enzymes to play a structural as well as a glycolytic role suggests that the presence of enolase at the centrosome may be correlated with the organization of both the interphase cytoskeleton and the mitotic spindle.