Thousands Of Merozoites Research Articles

A Micronemal Protein, Scot1, Is Essential for Apicoplast Biogenesis and Liver Stage Development in Plasmodium berghei.

Plasmodium sporozoites invade hepatocytes, transform into liver stages, and replicate into thousands of merozoites that infect erythrocytes and cause malaria. Proteins secreted from micronemes play an essential role in hepatocyte invasion, and unneeded micronemes are subsequently discarded for replication. The liver-stage parasites are potent immunogens that prevent malarial infection. Late liver stage-arresting genetically attenuated parasites (GAPs) exhibit greater protective efficacy than early GAP. However, the number of late liver-stage GAPs for generating GAPs with multiple gene deletions is limited. Here, we identified Scot1 (Sporozoite Conserved Orthologous Transcript 1), which was previously shown to be upregulated in sporozoites, and by endogenous tagging with mCherry, we demonstrated that it is expressed in the sporozoite and liver stages in micronemes. Using targeted gene deletion in Plasmodium berghei, we showed that Scot1 is essential for late liver-stage development. Scot1 KO sporozoites grew normally into liver stages but failed to initiate blood-stage infection in mice due to impaired apicoplast biogenesis and merozoite formation. Bioinformatic studies suggested that Scot1 is a metal-small-molecule carrier protein. Remarkably, supplementation with metals in the culture of infected Scot1 KO cells did not rescue their phenotype. Immunization with Scot1 KO sporozoites in C57BL/6 mice confers protection against malaria via infection. These proof-of-concept studies will enable the generation of P. falciparum Scot1 mutants that could be exploited to generate GAP malaria vaccines.

Same, same but different: Exploring Plasmodium cell division during liver stage development.

Plasmodium parasites have a complex life cycle alternating between a mosquito and a vertebrate host. Following the bite of an Anopheles female mosquito, Plasmodium sporozoites are transmitted from the skin to the liver; their first place of replication within the host. Successfully invaded sporozoites undergo a massive replication and growth involving asynchronous DNA replication and division that results in the generation of tens of thousands or even hundreds of thousands of merozoites depending on the Plasmodium species. The generation of a high number of daughter parasites requires biogenesis and segregation of organelles to finally reach a relatively synchronous cytokinesis event. At the end of liver stage (LS) development, merozoites are packed into merosomes and released into the bloodstream. They are then liberated and infect red blood cells to again produce merozoites by schizogony for the erythrocytic stage of the life cycle. Although parasite LS and asexual blood stage (ABS) differ in many respects, important similarities exist between the two. This review focuses on the cell division of Plasmodium parasite LS in comparison with other life cycle stages especially the parasite blood stage.

A genome-wide CRISPR-Cas9 screen identifies CENPJ as a host regulator of altered microtubule organization during Plasmodium liver infection

Prior to initiating symptomatic malaria, a single Plasmodium sporozoite infects a hepatocyte and develops into thousands of merozoites, in part by scavenging host resources, likely delivered by vesicles. Here, we demonstrate that host microtubules (MTs) dynamically reorganize around the developing liver stage (LS) parasite to facilitate vesicular transport to the parasite. Using a genome-wide CRISPR-Cas9 screen, we identified host regulators of cytoskeleton organization, vesicle trafficking, and ER/Golgi stress that regulate LS development. Foci of γ-tubulin localized to the parasite periphery; depletion of centromere protein J (CENPJ), a novel regulator identified in the screen, exacerbated this re-localization and increased infection. We demonstrate that the Golgi acts as a non-centrosomal MT organizing center (ncMTOC) by positioning γ-tubulin and stimulating MT nucleation at parasite periphery. Together, these data support a model where the Plasmodium LS recruits host Golgi to form MT-mediated conduits along which host organelles are recruited to PVM and support parasite development.

Plasmodium sporozoite phospholipid scramblase interacts with mammalian carbamoyl-phosphate synthetase 1 to infect hepatocytes

After inoculation by the bite of an infected mosquito, Plasmodium sporozoites enter the blood stream and infect the liver, where each infected cell produces thousands of merozoites. These in turn, infect red blood cells and cause malaria symptoms. To initiate a productive infection, sporozoites must exit the circulation by traversing the blood lining of the liver vessels after which they infect hepatocytes with unique specificity. We screened a phage display library for peptides that structurally mimic (mimotope) a sporozoite ligand for hepatocyte recognition. We identified HP1 (hepatocyte-binding peptide 1) that mimics a ~50 kDa sporozoite ligand (identified as phospholipid scramblase). Further, we show that HP1 interacts with a ~160 kDa hepatocyte membrane putative receptor (identified as carbamoyl-phosphate synthetase 1). Importantly, immunization of mice with the HP1 peptide partially protects them from infection by the rodent parasite P. berghei. Moreover, an antibody to the HP1 mimotope inhibits human parasite P. falciparum infection of human hepatocytes in culture. The sporozoite ligand for hepatocyte invasion is a potential novel pre-erythrocytic vaccine candidate.

Glyoxalase pathway is required for normal liver-stage proliferation of Plasmodium berghei

The glyoxalase system is a ubiquitous detoxification pathway of methylglyoxal, a cytotoxic byproduct of glycolysis. Actively proliferating cells, such as cancer cells, depend on their energy metabolism for glycolysis. Therefore, the glyoxalase system has been evaluated as a target of anticancer drugs. The malaria sporozoite, which is the infective stage of the malaria parasite, actively proliferates and produces thousands of merozoites within 2–3 days in hepatocytes. This is the first step of infection in mammalian hosts. The glyoxalase system appears to play an important role in this active proliferation stage of the malaria parasite in hepatocytes. In this study, we aimed to dissect the role of the glyoxalase system in malaria parasite proliferation in hepatocytes to examine its potential as a target of malaria prevention using a reverse genetics approach. The malaria parasite possesses a glyoxalase system, comprised of glyoxalases and GloI-like protein, in the cytosol and apicoplast. We generated cytosolic glyoxalase II (cgloII) knockout, apicoplast targeted glyoxalase gloII (tgloII) knockout, and cgloII and tgloII double-knockout parasites and performed their phenotypic analysis. We did not observe any defects in the cgloII or tgloII knockout parasites. In contrast, we observed approximately 90% inhibition of the liver-stage proliferation of cgloII and tgloII double-knockout parasites in vivo. These findings suggest that although the glyoxalase system is dispensable, it plays an important role in parasite proliferation in hepatocytes. Additionally, the results indicate a complementary relationship between the cytosolic and apicoplast glyoxalase pathways. We expect that the parasite utilizes a system similar to that observed in cancer cells to enable its rapid proliferation in hepatocytes; this process could be targeted in the development of novel strategies to prevent malaria.

Preparing for Transmission: Gene Regulation in Plasmodium Sporozoites

Plasmodium sporozoites are transmitted to mammals by anopheline mosquitoes and first infect the liver, where they transform into replicative exoerythrocytic forms, which subsequently release thousands of merozoites that invade erythrocytes and initiate the malaria disease. In some species, sporozoites can transform into dormant hypnozoites in the liver, which cause malaria relapses upon reactivation. Transmission from the insect vector to a mammalian host is a critical step of the parasite life cycle, and requires tightly regulated gene expression. Sporozoites are formed inside oocysts in the mosquito midgut and become fully infectious after colonization of the insect salivary glands, where they remain quiescent until transmission. Parasite maturation into infectious sporozoites is associated with reprogramming of the sporozoite transcriptome and proteome, which depends on multiple layers of transcriptional and post-transcriptional regulatory mechanisms. An emerging scheme is that gene expression in Plasmodium sporozoites is controlled by alternating waves of transcription activity and translational repression, which shape the parasite RNA and protein repertoires for successful transition from the mosquito vector to the mammalian host.

Protective efficacy and safety of liver stage attenuated malaria parasites.

During the clinically silent liver stage of a Plasmodium infection the parasite replicates from a single sporozoite into thousands of merozoites. Infection of humans and rodents with large numbers of sporozoites that arrest their development within the liver can cause sterile protection from subsequent infections. Disruption of genes essential for liver stage development of rodent malaria parasites has yielded a number of attenuated parasite strains. A key question to this end is how increased attenuation relates to vaccine efficacy. Here, we generated rodent malaria parasite lines that arrest during liver stage development and probed the impact of multiple gene deletions on attenuation and protective efficacy. In contrast to P. berghei strain ANKA LISP2(–) or uis3(–) single knockout parasites, which occasionally caused breakthrough infections, the double mutant lacking both genes was completely attenuated even when high numbers of sporozoites were administered. However, different vaccination protocols showed that LISP2(–) parasites protected better than uis3(–) and double mutants. Hence, deletion of several genes can yield increased safety but might come at the cost of protective efficacy.

Malaria and the liver: immunological hide-and-seek or subversion of immunity from within?

During the pre-erythrocytic asymptomatic phase of malarial infection, sporozoites develop transiently inside less than 100 hepatocytes that subsequently release thousands of merozoites. Killing of these hepatocytes by cytotoxic T cells (CTLs) confers protection to subsequent malarial infection, suggesting that this bottleneck phase in the parasite life cycle can be targeted by vaccination. During natural transmission, although some CTLs are generated in the skin draining lymph nodes, they are unable to eliminate the parasite, suggesting that the liver is important for the sporozoite to escape immune surveillance. The contribution of the organ to this process is unclear. Based on the known ability of several hepatic antigen-presenting cells (APCs) to induce primary activation of CD8 T cells and tolerance, malarial antigens presented by both infected hepatocytes and/or hepatic cross-presenting APCs should result in tolerance. However, our latest model predicts that due to the low frequency of infected hepatocytes, some T cells recognizing sporozoite epitopes with high affinity should differentiate into CTLs. In this review, we discuss two possible models to explain why CTLs generated in the liver and skin draining lymph nodes are unable to eliminate the parasite: (1) sporozoites harness the tolerogenic property of the liver; (2) CTLs are not tolerized but fail to detect infected cells due to sparse infection of hepatocytes and the very short liver stage. We propose that while malaria sporozoites might use the ability of the liver to tolerize both naive and effector cells, they have also developed strategies to decrease the probability of encounter between CTLs and infected liver cells. Thus, we predict that to achieve protection, vaccination strategies should aim to boost intrahepatic activation and/or increase the chance of encounter between sporozoite-specific CTLs and infected hepatocytes.

In vitro alterations do not reflect a requirement for host cell cycle progression during Plasmodium liver stage infection.

Prior to invading nonreplicative erythrocytes, Plasmodium parasites undergo their first obligate step in the mammalian host inside hepatocytes, where each sporozoite replicates to generate thousands of merozoites. While normally quiescent, hepatocytes retain proliferative capacity and can readily reenter the cell cycle in response to diverse stimuli. Many intracellular pathogens, including protozoan parasites, manipulate the cell cycle progression of their host cells for their own benefit, but it is not known whether the hepatocyte cell cycle plays a role during Plasmodium liver stage infection. Here, we show that Plasmodium parasites can be observed in mitotic hepatoma cells throughout liver stage development, where they initially reduce the likelihood of mitosis and ultimately lead to significant acquisition of a binucleate phenotype. However, hepatoma cells pharmacologically arrested in S phase still support robust and complete Plasmodium liver stage development, which thus does not require cell cycle progression in the infected cell in vitro. Furthermore, murine hepatocytes remain quiescent throughout in vivo infection with either Plasmodium berghei or Plasmodium yoelii, as do Plasmodium falciparum-infected primary human hepatocytes, demonstrating that the rapid and prodigious growth of liver stage parasites is accomplished independent of host hepatocyte cell cycle progression during natural infection.

Signatures of malaria vaccine efficacy in ageing murine immune memory

Malaria transmission occurs by mosquito bite. Thereafter, Plasmodium sporozoites specifically invade the liver, where they develop into thousands of merozoites that initiate blood-stage infection and clinical malaria. The pre-erythrocytic phase of a Plasmodium infection is the target of experimental whole-parasite vaccines against malaria. Repeated immunizations with high doses of live, metabolically active sporozoites can induce protracted protection against Plasmodium reinfection. Parasites lacking a Plasmodium-specific apicoplast protein, termed PALM, arrest very late during intrahepatic development just prior to liver merozoite release and can elicit sterile protection with two immunization doses only. In this report, we show in the robust Plasmodium berghei-C57BL/6 model that partial protection extends beyond 1 year after the last immunization. In ageing mice, intracellular cytokine staining of Plasmodium peptide-stimulated intrahepatic CD8+ T cells revealed elevated levels of interferon gamma in vaccinated mice. We conclude that antigen-specific T cells persist in the target organ and are critical signatures of lasting protection. Our data also support the notions that memory T-cell responses generated early in life remain largely intact well into old age and that murine Plasmodium vaccination and infection models are suitable to study the mechanisms of maintenance and efficiency of adaptive immunity during immunosenescence.

Atomic Force Microscopy for the Characterisation of the Effects and Treatment of Infectious Parasites

In this talk the utility of atomic force microscopy (AFM) for research into infectious parasites will be discussed. AFM has grown from relatively recent beginnings to become an extremely powerful technique in the life sciences, coupling high resolution imaging with a range of non-imaging experiments. Importantly, these experiments can be performed in situ, even on individual molecules or on live cells.The two examples discussed relate to the important diseases leishmaniasis and malaria. Leishmaniasis is a disease caused by the protozoan parasite of the Leishmania genera, and causes approximately 60,000 deaths per year. Despite the high death toll, the disease has been the subject of relatively little research and little treatment is available, probably because the most severe cases are confined to developing nations. The most severe form, visceral leishmaniasis is caused by the species known as Leishmania infantum (syn. L. chagasi). A promising new anti-leishmania drug, DS01 has been recently isolated from amphibian secretions and can kill L. infantum in low concentrations. We were able to culture and prepare for microscopy L. infantum promastigotes for the first time, as well as to study the effects of DS01 on cell morphology and membrane integrity. The results from both AFM and SEM are highly complementary and illustrate the possibility of membrane-focussed activity as well as the possibility of attack on the flagella (figure 1).Malaria is one of the most deadly diseases in the world, killing more than 600,000 people per year, mostly in low-income countries. It is caused by Plasmodium parasites, and the most commonly studied stage is that in which the parasite invades the blood. Prior to blood invasion, the parasites infect hepatocytes in the liver, with formation of a parasitophorous vacuole, where they develop into exoerythrocytic forms and multiply to generate thousands of merozoites, later released into the bloodstream and causing disease. However, infection of liver cells, which is clinically silent, is required for disease progression. We studied infection of liver cells by Plasmodium using combined epifluorescence and atomic force microscopy. We observed significant changes in cell morphology as infection progressed (figure 2). Furthermore we made nanoindentation measurements with the AFM, to determine cellular stiffness. We observed stiffening of the cells after 48 hours of infection compared to uninfected cells. This was a cellular response to the Plasmodium infection, rather than a result of the stiffness of the invading parasites themselves. This stiffening may be caused by reinforcement of cytoskeletal structures, and we believe this may reflect a self-defence mechanism by the cell itself.

Adaptive clinical trials of three PfSPZ products for development of a whole sporozoite vaccine that prevents Plasmodium falciparum infection, disease and transmission

An ideal, single stage vaccine useful for elimination of Plasmodium falciparum (Pf) would prevent infection at the pre-erythrocytic stage of the parasite life cycle, thereby preventing all Pf-caused disease and transmission from humans to mosquitoes. The only approach to immunization shown to consistently induce greater than 90% protection against infection and protection sustained for at least 10-28 months has been immunization by mosquito bite with whole Pf sporozoites (SPZ) of two types. The first type, radiation-attenuated PfSPZ, invade hepatoctyes and expresses new proteins, but cannot replicate. The second type fully develop in hepatocytes, producing tens of thousands of merozoites that invade erythrocytes, but are unable to fully develop within erythrocytes because they are killed by an antimalarial drug. This approach called chemoprophylaxis with sporozoites (CPS) harnesses the infectious agent’s inherent replicative properties to amplify production of protective immunogens spanning multiple developmental stages, and then eliminates the infectious agent with an anti-infective drug before the onset of disease. Sanaria was founded to develop PfSPZ vaccines. The first vaccine developed and tested was the PfSPZ Vaccine. The PfSPZ Vaccine is comprised of aseptic, purified, radiation attenuated, cryopreserved PfSPZ. It was shown to be safe and well-tolerated when administered ID or SC to 80 volunteers in the U.S., but sub-optimally immunogenic. It is now being tested in the U.S. and soon in Tanzania when administered by IV injection, since it induced high levels of PfSPZ-specific CD8+ T cells in the livers of immunized non-human primates when administered IV. A second product, PfSPZ Challenge, is comprised of non-irradiated, fully infectious PfSPZ. PfSPZ Challenge has been shown to infect 100% of volunteers after ID or IM administration by needle and syringe. It has been or will be tested for optimization of adminstration by the ID, IM, and IV routes in 2012 or early 2013 in the Netherlands, UK, Tanzania, U.S., Germany, Spain, and Kenya. A third product, PfSPZ-CVac, is comprised of PfSPZ Challenge administered to volunteers receiving chloroquine chemoprophylaxis. It will be assessed in 2012-2013 in the Netherlands, Mali, Germany and Tanzania. Assessment of these three products in synergistic, interactive and adaptive clinical trials will facilitate progress toward optimizing administration and dosage regimen of all three whole PfSPZ products, as well as those developed in the future from genetically altered parasites, thereby facilitating licensure of one or more PfSPZ-based vaccines. Progress and plans for development will be discussed.

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.

Nanofiber-Based Expansion and Differentiation Technology for High-Volume Ex Vivo Production of Reticulocytes for P. Vivax Malaria Research

Abstract 2357Infection by Plasmodium Vivax (P. Vivax) is the most common cause of Sleeping Malaria. P. Vivax and other plasmodia have grown increasingly resistant to antimalarial drugs. Introduced by mosquito bite, P. vivax sporozoites enter circulation and preferentially penetrate reticulocytes by attaching to the Fya and Fyb Duffy antigen/chemokine receptor (DARC) via PvRBP-1 and PvRBP-2 proteins located at their apical poles. Once in a reticulocyte, the parasite begins to reproduce asexually, releasing of thousands of merozoites into circulation. At this point, merozoites can also enter the liver and triggering relapses months or years later.The emergence of drug-resistant strains of p. vivax has stimulated development of new vaccines and treatments, but progress has been slowed by the dearth of reliable screening platforms. Many vaccine candidates have been developed to act upon vivax merozoites by preventing binding of PvRBP-1 and 2 to DARC, thereby arresting reproduction. However, there is a distinct lack of in vitro models to evaluate candidates that employ this mechanism. We are addressing this issue with a novel ex vivo expansion and differentiation technology for large-scale production of DARC expressing reticulocytes for in vitro P. vivax infection studies.This technology comprises an expansion system that can produce high yields of hematopoietic precursors (CD133+/CD34+ cells) from a variety of sources (marrow, peripheral blood, and cord blood), and a differentiation system to produce a relatively pure population of enucleated erythrocytes. In this study, we have refined the polyethersulfone (PES) nanofiber-based culturing system containing growth factors and cytokines in a serum-free media, to expand hematopoietic stem and progenitor cells (HSPC) ex vivo. This expansion technology allows rapid 200-fold ex vivo proliferation within 7 days of umbilical cord blood derived CD133+/CD34+ HSPCs from a DARC+ donor.Following expansion, over 50% of these cells retained HSPC phenotype (expression of CD34+). We have subsequently demonstrated that feeder layer free three-step differentiation of nanofiber-expanded cells using cytokines results in a population containing predominately enucleated reticulocyte-like cells. At 21 days of differentiation, cells had expanded 50-fold. Around 41% of cells were enucleated reticulocytes. These cells expressed glycophorin-A, a major sialoglycoprotein present on the human erythrocyte membrane. ∼28% of cells were CD36+, and ∼70% were CD71+ indicating an erythroid lineage.These results suggest that this technology can produce a population of DARC+ reticulocytes that is ∼5,000-fold greater than the starting population of HSPCs. We are partnering with leading malaria vaccine researchers to demonstrate that these reticulocytes can be parasitized by p. vivax. We believe that this will provide a unique platform to jumpstart research of malaria parasites and enable rapid development of effective vaccines. Further development of this technology may also have significant implications for large-scale ex vivo production of erythrocytes for general use. [Display omitted] Reticulocyte-like cells and expelled nuclei during differentiation of nanofiber-expanded HSPC. Disclosures:No relevant conflicts of interest to declare.

Superinfection in malaria: Plasmodium shows its iron will

After the bite of a malaria-infected mosquito, the Plasmodium sporozoite infects liver cells and produces thousands of merozoites, which then infect red blood cells, causing malaria. In malaria-endemic areas, several hundred infected mosquitoes can bite an individual each year, increasing the risk of superinfection. However, in infants that are yet to acquire immunity, superinfections are infrequent. We have recently shown that blood-stage parasitaemia, above a minimum threshold, impairs the growth of a subsequent sporozoite infection of liver cells. Blood-stage parasites stimulate the production of the host iron-regulatory factor hepcidin, which redistributes iron away from hepatocytes, reducing the development of the iron-dependent liver stage. This could explain why Plasmodium superinfection is not often found in young nonimmune children. Here, we discuss the impact that such protection from superinfection might have in epidemiological settings or in programmes for controlling malaria, as well as how the induction of hepcidin and redistribution of iron might influence anaemia and the outcome of non-Plasmodium co-infections.

APPLICATION OF MALARIA DETECTION OF DRAWING BLOOD CELLS USING MICROSCOPIC OpenCV

The goal of the research is to produce an application, which can detect malaria on patient through microscopic digital image of blood sample. The research methods are data collection, design analysis, testing and evaluation. The used application methods are image pre-processing, morphology and image segmentation using OpenCV. The expected result is a creation of application, which can be able to detect malaria on a microscopic digital image of patient blood sample. The conclusion is that the application can detect malaria from young trophozoites stadium and gametesocytes from the picture.Keywords: Detection; Malaria; Computer Vision; OpenCVINTRODUCTIONSystem technology of computer-based with artificial intelligence already can be used in medicine field, for example, to resolve the problems: detecting specific disease and its symptoms, analyzing the content of a sample, monitoring the condition of an organ, and others. Nevertheless, the medical field is very wide, so for detecting diseases problems, not yet much disease that detection can be done with a computer-based system. One example of the issues is well-known disease detection, which is malaria. Malaria is classified as a serious disease because it can cause death if it is not treated properly. Malaria has various types and can affect anyone anywhere. The symptoms of malaria is really common as it may appear in daily life, but cannot always indicate that a person infected with malaria. Indications, which can show that a person infected with malaria, are the clinical examination and blood tests.With the blood test, the treatment of malaria can be implemented correctly and precisely. It needs technology that can detect malaria correctly and precisely. The solution is the method of support vector machine that can detect malaria in humans by viewing image of appearance blood cells.METHODThe methods used in this research are data collection, analysis and design. The data collection includes literature studies about computer division with OpenCV and data collection of microscopic of blood sample. Analysis method includes process, detection procedure and malaria diagnosis. While design method includes steps of detection implementation and diagnosis to the application program, coding and continued with evaluation.MalariaMalaria parasites in human have a life cycle that requires a human host and mosquito host. In the anopheles mosquito, plasmodium does sexual reproduction. In humans, these parasites asexual reproduction, starting in the liver cells (hepatocytes), then repeatedly in the red blood cells (erythrocytes).While an infected female anopheles mosquito is sucking human’s blood, at the same time the mosquito inserts its saliva that is to keep the capillary vessels, which is inhaled not forming a blood clots factor that causes the blood flow stops. At this time the parasite creates sporozoites to enter the blood flow and infect hepatocytes. For one until two weeks (depends on plasmodium species), each sporozoites creates schizont; a structure that contains thousands of merozoites. When schizont is mature, hepatocytes will rupture and release merozoites to blood flow.In plasmodium vivax and plasmodium ovale, sporozoites develops into hipnozoit; a form of plasmodium that in dorman phase during several months to years. When hipnozoit re-activate, they will evolve into schizont that will cause recurrent symptoms to the infected person.Next is the merozoites, which is released to the blood flow, will invade erythrocyte then they will grow and consume hemoglobin. In erythrocyte, half of merozoites will grow to another phase of asexual, which creates schizont filled with merozoites. When schizont is mature, the cell will rupture and merozoites will be released and invade erythrocyte.

Plasmodium falciparum liver stage antigen-1 is cross-linked by tissue transglutaminase

BackgroundPlasmodium falciparum sporozoites injected by mosquitoes into the blood rapidly enter liver hepatocytes and undergo pre-erythrocytic developmental schizogony forming tens of thousands of merozoites per hepatocyte. Shortly after hepatocyte invasion, the parasite starts to produce Liver Stage Antigen-1 (LSA-1), which accumulates within the parasitophorous vacuole surrounding the mass of developing merozoites. The LSA-1 protein has been described as a flocculent mass, but its role in parasite development has not been determined.MethodsRecombinant N-terminal, C-terminal or a construct containing both the N- and C- terminal regions flanking two 17 amino acid residue central repeat sequences (LSA-NRC) were subjected to in vitro modification by tissue transglutaminase-2 (TG2) to determine if cross-linking occurred. In addition, tissue sections of P. falciparum-infected human hepatocytes were probed with monoclonal antibodies to the isopeptide ε-(γ-glutamyl)lysine cross-bridge formed by TG2 enzymatic activity to determine if these antibodies co-localized with antibodies to LSA-1 in the growing liver schizonts.ResultsThis study identified a substrate motif for (TG2) and a putative casein kinase 2 phosphorylation site within the central repeat region of LSA-1. The function of TG2 is the post-translational modification of proteins by the formation of a unique isopeptide ε-(γ-glutamyl)lysine cross-bridge between glutamine and lysine residues. When recombinant LSA-1 protein was crosslinked in vitro by purified TG2 in a calcium dependent reaction, a flocculent mass of protein was formed that was highly resistant to degradation. The cross-linking was not detectably affected by phosphorylation with plasmodial CK2 in vitro. Monoclonal antibodies specific to the very unique TG2 catalyzed ε- lysine cross-bridge co-localized with antibodies to LSA-1 in infected human hepatocytes providing visual evidence that LSA-1 was cross-linked in vivo.ConclusionsWhile the role of LSA-1 is still unknown these results suggest that it becomes highly cross-linked which may aid in the protection of the parasite as it develops.

Establishment of an In Vitro Assay for Assessing the Effects of Drugs on the Liver Stages of Plasmodium vivax Malaria

Plasmodium vivax (Pv) is the second most important human malaria parasite. Recent data indicate that the impact of Pv malaria on the health and economies of the developing world has been dramatically underestimated. Pv has a unique feature in its life cycle. Uninucleate sporozoites (spz), after invasion of human hepatocytes, either proceed to develop into tens of thousands of merozoites within the infected hepatocytes or remain as dormant forms called hypnozoites, which cause relapses of malaria months to several years after the primary infection. Elimination of malaria caused by Pv will be facilitated by developing a safe, highly effective drug that eliminates Pv liver stages, including hypnozoites. Identification and development of such a drug would be facilitated by the development of a medium to high throughput assay for screening drugs against Pv liver stages. We undertook the present pilot study to (1) assess the feasibility of producing large quantities of purified, vialed, cryopreserved Pv sporozoites and (2) establish a system for culturing the liver stages of Pv in order to assess the effects of drugs on the liver stages of Pv. We used primaquine (PQ) to establish this assay model, because PQ is the only licensed drug known to clear all Pv hepatocyte stages, including hypnozoites, and the effect of PQ on Pv hepatocyte stage development in vitro has not previously been reported. We report that we have established the capacity to reproducibly infect hepatoma cells with purified, cyropreserved Pv spz from the same lot, quantitate the primary outcome variable of infected hepatoma cells and demonstrate the inhibitory activity of primaquine on the infected hepatoma cells. We have also identified small parasite forms that may be hypnozoites. These data provide the foundation for finalizing a medium throughput, high content assay to identify new drugs for the elimination of all Pv liver stages.

Identification and Characterization of a Liver Stage-Specific Promoter Region of the Malaria Parasite Plasmodium

During the blood meal of a Plasmodium-infected mosquito, 10 to 100 parasites are inoculated into the skin and a proportion of these migrate via the bloodstream to the liver where they infect hepatocytes. The Plasmodium liver stage, despite its clinical silence, represents a highly promising target for antimalarial drug and vaccine approaches. Successfully invaded parasites undergo a massive proliferation in hepatocytes, producing thousands of merozoites that are transported into a blood vessel to infect red blood cells. To successfully develop from the liver stage into infective merozoites, a tight regulation of gene expression is needed. Although this is a very interesting aspect in the biology of Plasmodium, little is known about gene regulation in Plasmodium parasites in general and in the liver stage in particular. We have functionally analyzed a novel promoter region of the rodent parasite Plasmodium berghei that is exclusively active during the liver stage of the parasite. To prove stage-specific activity of the promoter, GFP and luciferase reporter assays have been successfully established, allowing both qualitative and accurate quantitative analysis. To further characterize the promoter region, the transcription start site was mapped by rapid amplification of cDNA ends (5′-RACE). Using promoter truncation experiments and site-directed mutagenesis within potential transcription factor binding sites, we suggest that the minimal promoter contains more than one binding site for the recently identified parasite-specific ApiAP2 transcription factors. The identification of a liver stage-specific promoter in P. berghei confirms that the parasite is able to tightly regulate gene expression during its life cycle. The identified promoter region might now be used to study the biology of the Plasmodium liver stage, which has thus far proven problematic on a molecular level. Stage-specific expression of dominant-negative mutant proteins and overexpression of proteins normally active in other life cycle stages will help to understand the function of the proteins investigated.

A novel type of Plasmodium sporozoite transcellular migration

During liver-stage malaria infection, Plasmodium sporozoites must invade a hepatocyte in order to produce thousands of merozoites, the stage that infects erythrocytes and causes malarial pathology. One of the first steps of hepatocyte infection is the invagination of the host cell membrane, which shelters the parasite within a parasitophorous vacuole (PV) and allows parasite development inside the host cell. Sporozoites also have another peculiar type of cell invasion, in which the parasite breaches the host cell plasma membrane and transmigrates, free in the cytoplasm, through the host cell. This cell wounding and crossing process is termed cell traversal. Here, using live-cell imaging techniques, fluorescent markers of the host cell plasma membrane and a mutant parasite lacking cell traversal activity, we describe a new type of sporozoite transcellular migration. We have observed wild-type Plasmodium berhgei sporozoites moving through hepatocytes while surrounded by host cell membrane. Additionally, a small percentage of sporozoites drag host cell membrane with them when they leave hepatocytes, forming aggregates of membrane we have termed membrane bobbins. Membrane-surrounded migrating sporozoites and membrane bobbins are also observed during transmigration of primary liver sinusoidal endothelial cells. As cell traversal deficient sporozoites are still able to move across the cell surrounded by host cell plasma membrane, we define this new transcellular migration as being distinct from cell traversal. We hypothesize that this form of transmigration starts with the PV membrane formation similar to invasion with the exception that these sporozoites continue migration, ultimately leaving the cell by membrane fusion or disruption of the vacuole membrane and the plasma membrane.