Exoerythrocytic Stages Research Articles

Description, molecular characterisation, diagnostics and life cycle of Plasmodium elongatum (lineage pERIRUB01), the virulent avian malaria parasite

Plasmodium elongatum causes severe avian malaria and is distributed worldwide. This parasite is of particular importance due to its ability to develop and cause lethal malaria not only in natural hosts, but also in non-adapted endemic birds such as the brown kiwi and different species of penguins. Information on vectors of this infection is available but is contradictory. PCR-based analysis indicated the possible existence of a cluster of closely related P. elongatum lineages which might differ in their ability to develop in certain mosquitoes and birds. This experimental study provides information about molecular and morphological characterisation of a virulent P. elongatum strain (lineage pERIRUB01) isolated from a naturally infected European robin, Erithacus rubecula. Phylogenetic analysis based on partial cytochrome b gene sequences showed that this parasite lineage is closely related to P. elongatum (lineage pGRW6). Blood stages of both parasite lineages are indistinguishable, indicating that they belong to the same species. Both pathogens develop in experimentally infected canaries, Serinus canaria, causing death of the hosts. In both these lineages, trophozoites and erythrocytic meronts develop in polychromatic erythrocytes and erythroblasts, gametocytes parasitize mature erythrocytes, exoerythrocytic stages develop in cells of the erythrocytic series in bone marrow and are occasionally reported in spleen and liver. Massive infestation of bone marrow cells is the main reason for bird mortality. We report here on syncytium-like remnants of tissue meronts, which slip out of the bone marrow into the peripheral circulation, providing evidence that the syncytia can be a template for PCR amplification. This finding contributes to better understanding positive PCR amplifications in birds when parasitemia is invisible and improved diagnostics of abortive haemosporidian infections. Sporogony of P. elongatum (pERIRUB01) completes the cycle and sporozoites develop in widespread Culex quinquefasciatus and Culex pipiens pipiens form molestus mosquitoes. This experimental study provides information on virulence and within species lineage diversity in a single pathogenic species of haemosporidian parasite.

Identification and Characterization of the Rhoptry Neck Protein 2 in Babesia divergens and B. microti.

Apicomplexan parasites include those of the genera Plasmodium, Cryptosporidium, and Toxoplasma and those of the relatively understudied zoonotic genus Babesia In humans, babesiosis, particularly transfusion-transmitted babesiosis, has been emerging as a major threat to public health. Like malaria, the disease pathology is a consequence of the parasitemia which develops through cyclical replication of Babesia parasites in host erythrocytes. However, there are no exoerythrocytic stages in Babesia, so targeting of the blood stage and associated proteins to directly prevent parasite invasion is the most desirable option for effective disease control. Especially promising among such molecules are the rhoptry neck proteins (RONs), whose homologs have been identified in many apicomplexan parasites. RONs are involved in the formation of the moving junction, along with AMA1, but no RON has been identified and characterized in any Babesia spp. Here we identify the RON2 proteins of Babesia divergens (BdRON2) and B. microti (BmRON2) and show that they are localized apically and that anti-BdRON2 antibodies are significant inhibitors of parasite invasion in vitro Neither protein is immunodominant, as both proteins react only marginally with sera from infected animals. Further characterization of the direct role of both BdRON2 and BmRON2 in parasite invasion is required, but knowledge of the level of conformity of RON2 proteins within the apicomplexan phylum, particularly that of the AMA1-RON2 complex at the moving junction, along with the availability of an animal model for B. microti studies, provides a key to target this complex with a goal of preventing the erythrocytic invasion of these parasites and to further our understanding of the role of these conserved ligands in invasion.

High-Throughput Luciferase-Based Assay for the Discovery of Therapeutics That Prevent Malaria.

In order to identify the most attractive starting points for drugs that can be used to prevent malaria, a diverse chemical space comprising tens of thousands to millions of small molecules may need to be examined. Achieving this throughput necessitates the development of efficient ultra-high-throughput screening methods. Here, we report the development and evaluation of a luciferase-based phenotypic screen of malaria exoerythrocytic-stage parasites optimized for a 1536-well format. This assay uses the exoerythrocytic stage of the rodent malaria parasite, Plasmodium berghei, and a human hepatoma cell line. We use this assay to evaluate several biased and unbiased compound libraries, including two small sets of molecules (400 and 89 compounds, respectively) with known activity against malaria erythrocytic-stage parasites and a set of 9886 diversity-oriented synthesis (DOS)-derived compounds. Of the compounds screened, we obtain hit rates of 12–13 and 0.6% in preselected and naïve libraries, respectively, and identify 52 compounds with exoerythrocytic-stage activity less than 1 μM and having minimal host cell toxicity. Our data demonstrate the ability of this method to identify compounds known to have causal prophylactic activity in both human and animal models of malaria, as well as novel compounds, including some exclusively active against parasite exoerythrocytic stages.

Exploring MIA-QSARs' for antimalarial quinolon-4(1H)-imines.

A series of quinolon-4(1H)-imines have been recently discovered as antimalarials, targeting both the exoerythrocytic and erythrocytic stages of the parasite's development stages, which correspond to the phase of clinical symptoms. Endowed with chemical and metabolic stability, the quinolon-4(1H)- imines are thus presented as promissory dual-stage antimalarials. Three versions of multivariate image analysis applied to quantitative structure-activity relationship (MIA-QSAR) methods, namely traditional MIA-QSAR, augmented MIA-QSAR (aug-MIA-QSAR) and color-encoded aug-MIA-QSAR (aug- MIA-QSARcolor), were applied to model the antimalarial activities in this series of compounds. The multiple linear regression models indicated that the aug-MIA-QSAR method is more predictive and reliable than the others (R(2) = 0.8079, R(2)cv = 0.6647 and R(2)pred = 0.9691) for this series of compounds. The selected aug- MIA-QSAR descriptors were used for pattern recognition using discriminant analysis by partial least squares (PLS-DA), in order to separate compounds with low, moderate and high bioactivities.

Priming of CD8(+) T Cell Responses to Liver Stage Malaria Parasite Antigens.

While the role of malaria parasite-specific memory CD8+ T cells in the control of exo-erythrocytic stages of malaria infection is well documented and generally accepted, a debate is still ongoing regarding both the identity of the anatomic site where the activation of naive pathogen-specific T cells is taking place and contribution of different antigen-presenting cells (APCs) into this process. Whereas some studies infer a role of professional APCs present in the lymph nodes draining the site of parasite injection by the mosquito, others argue in favor of the liver as a primary organ and hepatocytes as stimulators of naïve parasite-specific T cell responses. This review aims to critically analyze the current knowledge and outline new lines of research necessary to understand the induction of protective cellular immunity against the malaria parasite.

Description of the first cryptic avian malaria parasite, Plasmodium homocircumflexum n. sp., with experimental data on its virulence and development in avian hosts and mosquitoes

For over 100years studies on avian haemosporidian parasite species have relied on similarities in their morphology to establish a species concept. Some exceptional cases have also included information about the life cycle and sporogonic development. More than 50 avian Plasmodium spp. have now been described. However, PCR-based studies show a much broader diversity of haemosporidian parasites, indicating the possible existence of a diverse group of cryptic species. In the present study, using both similarity and phylogenetic species definition concepts, we believe that we report the first characterised cryptic speciation case of an avian Plasmodium parasite. We used sequence information on the mitochondrial cytochrome b gene and constructed phylogenies of identified Plasmodium spp. to define their position in the phylogenetic tree. After analysis of blood stages, the morphology of the parasite was shown to be identical to Plasmodium circumflexum. However, the geographic distribution of the new parasite, the phylogenetic information, as well as patterns of development of infection, indicate that this parasite differs from P. circumflexum. Plasmodium homocircumflexum n. sp. was described based on information about genetic differences from described lineages, phylogenetic position and biological characters. This parasite develops parasitemia in experimentally infected birds – the domestic canary Serinus canaria domestica, siskin Carduelis spinus and crossbill Loxia curvirostra. Anaemia caused by high parasitemia, as well as cerebral paralysis caused by exoerythrocytic stages in the brain, are the main reasons for mortality. Exoerythrocytic stages also form in other organs (heart, kidneys, liver, lungs, spleen, intestines and pectoral muscles). DNA amplification was unsuccessful from faecal samples of heavily infected birds. The sporogonic development initiates, but is abortive, at the oocyst stage in two common European mosquito species, Culex pipiens pipiens (forms pipiens and molestus) and Aedes vexans. Vectors of this Plasmodium sp. remain unknown.

The medicinal chemistry and drug development of novel antimalarials.

The medicinal chemistry and drug development of novel antimalarials.

Discovery of HDAC inhibitors with potent activity against multiple malaria parasite life cycle stages

In this work we investigated the antiplasmodial activity of a series of HDAC inhibitors containing an alkoxyamide connecting-unit linker region. HDAC inhibitor 1a (LMK235), previously shown to be a novel and specific inhibitor of human HDAC4 and 5, was used as a starting point to rapidly construct a mini-library of HDAC inhibitors using a straightforward solid-phase supported synthesis. Several of these novel HDAC inhibitors were found to have potent in vitro activity against asexual stage Plasmodium falciparum malaria parasites. Representative compounds were shown to hyperacetylate P. falciparum histones and to inhibit deacetylase activity of recombinant PfHDAC1 and P. falciparum nuclear extracts. All compounds were also screened in vitro for activity against Plasmodium berghei exo-erythrocytic stages and selected compounds were further tested against late stage (IV and V) P. falciparum gametocytes. Of note, some compounds showed nanomolar activity against all three life cycle stages tested (asexual, exo-erythrocytic and gametocyte stages) and several compounds displayed significantly increased parasite selectivity compared to the reference HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). These data suggest that it may be possible to develop HDAC inhibitors that target multiple malaria parasite life cycle stages.

Molecular characterization of five widespread avian haemosporidian parasites (Haemosporida), with perspectives on the PCR-based detection of haemosporidians in wildlife.

Haemosporidians (Haemosporida) are cosmopolitan in birds. Over 250 species of these blood parasites have been described and named; however, molecular markers remain unidentified for the great majority of them. This is unfortunate because linkage between DNA sequences and identifications based on morphological species can provide important information about patterns of transmission, virulence, and evolutionary biology of these organisms. There is an urgent need to remedy this because few experts possess the knowledge to identify haemosporidian species and few laboratories are involved in training these taxonomic skills. Here, we describe new mitochondrial cytochrome b markers for the polymerase chain reaction (PCR)-based detection of four widespread species of avian Haemoproteus (Haemoproteus hirundinis, Haemoproteus parabelopolskyi, Haemoproteus pastoris, Haemoproteus syrnii) and 1 species of Plasmodium (Plasmodium circumflexum). Illustrations of blood stages of the reported species are given, and morphological and phylogenetic analyses identify the DNA lineages that are associated with these parasites. This study indicates that morphological characters, which have been traditionally used in taxonomy of avian haemosporidian parasites, have a phylogenetic value. Perspectives on haemosporidian diagnostics using microscopic and PCR-based methods are discussed, particularly the difficulties in detection of light parasitemia, coinfections, and abortive parasite development. We emphasize that sensitive PCR amplifies more infections than can be transmitted; it should be used carefully in epidemiology studies, particularly in wildlife parasitology research. Because molecular studies are describing remarkably more parasite diversity than previously expected, the need for traditional taxonomy and traditional biological knowledge is becoming all the more crucial. The linkage of molecular and morphological approaches is worth more of the attention of researchers because this approach provides new knowledge for better understanding insufficiently investigated lethal diseases caused by haemosporidian infections, particularly on the exoerythrocytic (tissue) and vector stages. That requires close collaboration between researchers from different fields with a common interest.

The Malarial Serine Protease SUB1 Plays an Essential Role in Parasite Liver Stage Development

Transmission of the malaria parasite to its vertebrate host involves an obligatory exoerythrocytic stage in which extensive asexual replication of the parasite takes place in infected hepatocytes. The resulting liver schizont undergoes segmentation to produce thousands of daughter merozoites. These are released to initiate the blood stage life cycle, which causes all the pathology associated with the disease. Whilst elements of liver stage merozoite biology are similar to those in the much better-studied blood stage merozoites, little is known of the molecular players involved in liver stage merozoite production. To facilitate the study of liver stage biology we developed a strategy for the rapid production of complex conditional alleles by recombinase mediated engineering in Escherichia coli, which we used in combination with existing Plasmodium berghei deleter lines expressing Flp recombinase to study subtilisin-like protease 1 (SUB1), a conserved Plasmodium serine protease previously implicated in blood stage merozoite maturation and egress. We demonstrate that SUB1 is not required for the early stages of intrahepatic growth, but is essential for complete development of the liver stage schizont and for production of hepatic merozoites. Our results indicate that inhibitors of SUB1 could be used in prophylactic approaches to control or block the clinically silent pre-erythrocytic stage of the malaria parasite life cycle.

An establishment of exo-erythrocytic stage of malaria parasite for ex-vivo study of sporozoiticidals

An establishment of exo-erythrocytic stage of malaria parasite for ex-vivo study of sporozoiticidals

PFE0565w, a Plasmodium falciparum Protein Expressed in Salivary Gland Sporozoites

Because malaria is still a significant problem worldwide, additional control methods need to be developed. The Plasmodium sporozoite is a good target for control measures because it displays dual infectivity for both mosquito and vertebrate host tissues. The Plasmodium falciparum gene, PFE0565w, was chosen as a candidate for study based on data from PlasmoDB, the Plasmodium database, indicating that it is expressed both at the transcriptional and protein levels in sporozoites, likely encodes a putative surface protein, and may have a potential role in the invasion of host tissues. Additional sequence analysis shows that the PFE0565w protein has orthologs in other Plasmodium species, but none outside of the genus Plasmodium. PFE0565w expresses transcript during both the sporozoite and erythrocytic stages of the parasite life cycle, where an alternative transcript was discovered during the erythrocytic stages. Data show that transcript is not present during axenic exoerythrocytic stages. Despite transcript being present in several life cycle stages, the PFE0565w protein is present only during the salivary gland sporozoite stage. Because the PFE0565w protein is present in salivary gland sporozoites, it could be a novel candidate for a pre-erythrocytic stage vaccine.

The painstaking discovery of the hidden face of the human plasmodia

The difficulties encountered in the discovery of the exoerythrocytic stage of asexual cycle of human plasmodia are described. These illustrate how deference towards scientific orthodoxy and a degree of reluctance to question and to criticise can delay advances in knowledge.

Pathogenic mechanisms in Plasmodium falciparum malaria

The most recognized pathogenic mechanisms of the infection with Plasmodium falciparum, during both the erythrocytic and exo-erythrocytic stages are presented. Vascular obstruction explained by the sequestration of parasitized red blood cells and erythrocyte rosetting, mediated by different endothelial ligands and receptors, in addition to the inflammatory processes induced by the presence of the parasite, are central aspects in the pathogenesis of malaria that explain the processes of damage, dysfunction and cell death in various organs. Alterations such as increased vascular permeability, hypoxia and anaerobic metabolism leading to localized lesions in organs such as brain and lung, as well as to a generalized acidotic state with multisystem failure can be explained by events such as the injury and destruction of erythrocytes, hepatocytes and endothelial cells, the loss of endothelial integrity, and the activation of cell damage and apoptosis promoters.

Transcript and protein expression profile of PF11_0394, a Plasmodium falciparum protein expressed in salivary gland sporozoites

BackgroundPlasmodium falciparum malaria is a significant problem around the world today, thus there is still a need for new control methods to be developed. Because the sporozoite displays dual infectivity for both the mosquito salivary glands and vertebrate host tissue, it is a good target for vaccine development.MethodsThe P. falciparum gene, PF11_0394, was chosen as a candidate for study due to its potential role in the invasion of host tissues. This gene, which was selected using a data mining approach from PlasmoDB, is expressed both at the transcriptional and protein levels in sporozoites and likely encodes a putative surface protein. Using reverse transcription-polymerase chain reaction (RT-PCR) and green fluorescent protein (GFP)-trafficking studies, a transcript and protein expression profile of PF11_0394 was determined.ResultsThe PF11_0394 protein has orthologs in other Plasmodium species and Apicomplexans, but none outside of the group Apicomplexa. PF11_0394 transcript was found to be present during both the sporozoite and erythrocytic stages of the parasite life cycle, but no transcript was detected during axenic exoerythrocytic stages. Despite the presence of transcript throughout several life cycle stages, the PF11_0394 protein was only detected in salivary gland sporozoites.ConclusionsPF11_0394 appears to be a protein uniquely detected in salivary gland sporozoites. Even though a specific function of PF11_0394 has not been determined in P. falciparum biology, it could be another candidate for a new vaccine.

Extrahepatic exoerythrocytic forms of rodent malaria parasites at the site of inoculation: clearance after immunization, susceptibility to primaquine, and contribution to blood-stage infection.

Plasmodium sporozoites are inoculated into the skin of the mammalian host as infected mosquitoes probe for blood. A proportion of the inoculum enters the bloodstream and goes to the liver, where the sporozoites invade hepatocytes and develop into the next life cycle stage, the exoerythrocytic, or liver, stage. Here, we show that a small fraction of the inoculum remains in the skin and begins to develop into exoerythrocytic forms that can persist for days. Skin exoerythrocytic forms were observed for both Plasmodium berghei and Plasmodium yoelii, two different rodent malaria parasites, suggesting that development in the skin of the mammalian host may be a common property of plasmodia. Our studies demonstrate that skin exoerythrocytic stages are susceptible to destruction in immunized mice, suggesting that their aberrant location does not protect them from the host's adaptive immune response. However, in contrast to their hepatic counterparts, they are not susceptible to primaquine. We took advantage of their resistance to primaquine to test whether they could initiate a blood-stage infection directly from the inoculation site, and our data indicate that these stages are not able to initiate malaria infection.

Chronicle of a death foretold:Plasmodiumliver stage parasites decide on the fate of the host cell

Protozoan parasites of the genus Plasmodium are the causative agents of malaria. Despite more than 100years of research, the complex life cycle of the parasite still bears many surprises and it is safe to say that understanding the biology of the pathogen will keep scientists busy for many years to come. Malaria research has mainly concentrated on the pathological blood stage of Plasmodium parasites, leaving us with many questions concerning parasite development within the mosquito and during the exo-erythrocytic stage in the vertebrate host. After the discovery of the Plasmodium liver stage in the middle of the last century, it remained understudied for many years but the realization that it represents a promising target for vaccination approaches has brought it back into focus. The last decade saw many new and exciting discoveries concerning the exo-erythrocytic stage and in this review we will discuss the highlights of the latest developments in the field.

Hemoglobin S and C Heterozygosity Enhances Neither the Magnitude nor Breadth of Antibody Responses to a Diverse Array of Plasmodium falciparum Antigens

Heterozygous states of hemoglobin (Hb) A and HbS (HbAS, sickle-cell trait) or HbC (HbAC) protect against Plasmodium falciparum malaria by unclear mechanisms. Several studies suggest that HbAS and HbAC accelerate the acquisition of immunity to malaria, possibly by enhancing P. falciparum-specific antibody responses. We used a protein microarray representing 491 P. falciparum proteins expressed during exoerythrocytic and erythrocytic stages of the life cycle to test the hypothesis that HbAS and HbAC enhance the P. falciparum-specific IgG response compared with normal HbAA. Plasma samples were collected from Malian children aged 2-10 years before and after a 6-month malaria season and were probed against the microarray. Immunoglobulin G (IgG) profiles of children with HbAA (n = 106), HbAS (n = 15), and HbAC (n = 20) were compared. Although the magnitude and breadth of P. falciparum-specific IgG responses increased with age and from before to after the malaria season in each antigen category, Hb type did not independently predict significant differences in P. falciparum-specific IgG profiles. These data do not support the hypothesis that HbAS and HbAC protect against malaria by enhancing P. falciparum-specific antibody responses. It remains possible that HbAS and HbAC protect against malaria by enhancing antibody responses to antigens not studied here or through other immune mechanisms.

Challenges in Antimalarial Drug Discovery

Malaria is one of the most devastating diseases in the world, affecting almost 225 million people a year, and causing over 780,000 deaths, most of which are children under the age of 5 years. Following the recent call for the eradication of the disease, supported by the WHO, there has been increasing investment into antimalarial drug-discovery projects. These activities are aimed at generating the next generation of molecules focused on the treatment and transmission-blocking of Plasmodium falciparum and Plasmodium vivax endo- and exo-erythrocytic stages of the parasite. This article summarizes the current top-level thinking regarding the prosecution of such endeavors and the disease-specific considerations in project planning.

Transition of Plasmodium Sporozoites into Liver Stage-Like Forms Is Regulated by the RNA Binding Protein Pumilio

Many eukaryotic developmental and cell fate decisions that are effected post-transcriptionally involve RNA binding proteins as regulators of translation of key mRNAs. In malaria parasites (Plasmodium spp.), the development of round, non-motile and replicating exo-erythrocytic liver stage forms from slender, motile and cell-cycle arrested sporozoites is believed to depend on environmental changes experienced during the transmission of the parasite from the mosquito vector to the vertebrate host. Here we identify a Plasmodium member of the RNA binding protein family PUF as a key regulator of this transformation. In the absence of Pumilio-2 (Puf2) sporozoites initiate EEF development inside mosquito salivary glands independently of the normal transmission-associated environmental cues. Puf2- sporozoites exhibit genome-wide transcriptional changes that result in loss of gliding motility, cell traversal ability and reduction in infectivity, and, moreover, trigger metamorphosis typical of early Plasmodium intra-hepatic development. These data demonstrate that Puf2 is a key player in regulating sporozoite developmental control, and imply that transformation of salivary gland-resident sporozoites into liver stage-like parasites is regulated by a post-transcriptional mechanism.