Search

The simian parasite Plasmodium knowlesi causes severe and fatal malaria infections in humans, but the process of host cell remodelling that underpins the pathology of this zoonotic parasite is only poorly understood. We have used serial block-face scanning electron microscopy to explore the topography of P. knowlesi-infected red blood cells (RBCs) at different stages of asexual development. The parasite elaborates large flattened cisternae (Sinton Mulligan's clefts) and tubular vesicles in the host cell cytoplasm, as well as parasitophorous vacuole membrane bulges and blebs, and caveolar structures at the RBC membrane. Large invaginations of host RBC cytoplasm are formed early in development, both from classical cytostomal structures and from larger stabilised pores. Although degradation of haemoglobin is observed in multiple disconnected digestive vacuoles, the persistence of large invaginations during development suggests inefficient consumption of the host cell cytoplasm. The parasite eventually occupies ~40% of the host RBC volume, inducing a 20% increase in volume of the host RBC and an 11% decrease in the surface area to volume ratio, which collectively decreases the ability of the P. knowlesi-infected RBCs to enter small capillaries of a human erythrocyte microchannel analyser. Ektacytometry reveals a markedly decreased deformability, whereas correlative light microscopy/scanning electron microscopy and python-based skeleton analysis (Skan) reveal modifications to the surface of infected RBCs that underpin these physical changes. We show that P. knowlesi-infected RBCs are refractory to treatment with sorbitol lysis but are hypersensitive to hypotonic lysis. The observed physical changes in the host RBCs may underpin the pathology observed in patients infected with P. knowlesi., (© 2019 The Authors. Cellular Microbiology Published by John Wiley & Sons Ltd.)

Water buffalo (Bubalus bubalis) is important for the economy of Asia, South America and parts of Europe. Coccidiosis is an important cause of neonatal mortality in livestock, including buffalo. Of more than 12 species of Eimeria in buffalo, Eimeria bareillyi is the most pathogenic. There are uncertainties concerning its asexual and sexual development. During a previously reported outbreak of fatal enteritis associated with E. bareillyi in buffaloes in the Netherlands, sections of small intestine were re-evaluated histologically and by transmission electron microscopy (TEM) to seek details of endogenous development. Profuse asexual multiplication occurred in the jejunum and ileum. Light microscopic examination revealed that parasites divided in two (probably endodyogeny) or more organisms. There were two or more generations of morphologically different merozoites; some of these observations were confirmed by TEM. Details of gametogonic development, including oocyst wall formation are provided. Schizogonic and gametogonic development described in the present study can serve as a guide for differential diagnosis of Eimeria species in histological sections of intestines of buffaloes.

Apical organellar proteins in Plasmodium falciparum merozoites play important roles upon invasion. To date, dense granule, the least studied apical organelle, secretes parasite proteins across the parasitophorous vacuole membrane (PVM) to remodel the infected erythrocyte. Although this phenomenon is key to parasite growth and virulence, only five proteins so far have been identified as dense granule proteins. Further elucidation of dense granule molecule(s) is therefore required. P. falciparum Exported Protein (EXP) 1, previously reported as a parasitophorous vacuole membrane (PVM) protein, is considered essential for parasite growth. In this study, we characterized EXP1 using specific anti-EXP1 antibodies generated by immunization of wheat germ cell-free produced recombinant EXP1. Immunofluorescence microscopy (IFA) demonstrated that EXP1 co-localized with RESA, indicating that the protein is initially localized to dense granules in merozoites, followed by translocation to the PVM. The EXP1 localization in dense granule of merozoites and its translocation to the PVM after invasion of erythrocytes were further confirmed by immunoelectron microscopy. Here, we demonstrate that EXP1 is one of the dense granule proteins in merozoites, which is then transported to the PVM after invasion., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Plasmodium knowlesi, a zoonotic parasite causing severe-to-lethal malaria disease in humans, has only recently been adapted to continuous culture with human red blood cells (RBCs). In comparison with the most virulent human malaria, Plasmodium falciparum, there are, however, few cellular tools available to study its biology, in particular direct investigation of RBC invasion by blood-stage P. knowlesi merozoites. This leaves our current understanding of biological differences across pathogenic Plasmodium spp. incomplete. Here, we report a robust method for isolating viable and invasive P. knowlesi merozoites to high purity and yield. Using this approach, we present detailed comparative dissection of merozoite invasion (using a variety of microscopy platforms) and direct assessment of kinetic differences between knowlesi and falciparum merozoites. We go on to assess the inhibitory potential of molecules targeting discrete steps of invasion in either species via a quantitative invasion inhibition assay, identifying a class of polysulfonate polymer able to efficiently inhibit invasion in both, providing a foundation for pan-Plasmodium merozoite inhibitor development. Given the close evolutionary relationship between P. knowlesi and P. vivax, the second leading cause of malaria-related morbidity, this study paves the way for inter-specific dissection of invasion by all three major pathogenic malaria species.

A time-course study was conducted to resolve discrepancies in the literature and better define aspects of the Eimeria maxima life cycle such, as sites of development and both morphology and number of asexual stages. Broiler chickens were inoculated orally with five million E. maxima oocysts (APU1), and were necropsied at regular intervals from 12 to 120 h p.i. Small intestine tissue sections and smears were examined for developmental stages. The jejunum contained the highest numbers of developmental stages. At 12 h p.i., sporozoites were observed inside a parasitophorous vacuole (PV) in the epithelial villi and the lamina propria. By 24 h, sporozoites enclosed by a PV were observed in enterocytes of the glands of Lieberkühn. At 48 h p.i., sporozoites, elongated immature and mature schizonts, were all seen in the glands with merozoites budding off from a residual body. By 60 h, second-generation, sausage-shaped schizonts containing up to 12 merozoites were observed around a residual body in the villar tip of invaded enterocytes. At 72 and 96 h, profuse schizogony associated with third- and fourth-generation schizonts was observed throughout the villus. At 120 h, another generation (fifth) of schizonts were seen in villar tips as well as in subepithelium where gamonts and oocysts were also present; a few gamonts were in epithelium. Our finding of maximum parasitization of E. maxima in jejunum is important because this region is critical for nutrient absorption and weight gain.

The number of malaria vaccine candidates in preclinical and clinical development is limited. To identify novel blood-stage malaria vaccine candidates, we constructed a library of 1,827P. falciparum proteins prepared using the wheat germ cell-free system (WGCFS). Also, a high-throughput AlphaScreen procedure was developed to measure antibody reactivity to the recombinant products. Purified IgGs from residents in malaria endemic areas have shown functional activity against blood-stage parasites as judged by an in vitro parasite Growth Inhibition Assay (GIA). Therefore, we evaluated the GIA activity of 51 plasma samples prepared from Malian adults living in a malaria endemic area against the WGCFS library. Using the AlphaScreen-based immunoreactivity measurements, antibody reactivity against 3 proteins was positively associated with GIA activity. Since anti-LSA3-C responses showed the strongest correlation with GIA activity, this protein was investigated further. Anti-LSA3-C-specific antibody purified from Malian adult plasmas showed GIA activity, and expression of LSA3 in blood-stage parasites was confirmed by western blotting. Taken together, we identified LSA3 as a novel blood-stage vaccine candidate, and we propose that this system will be useful for future vaccine candidate discovery.

Over the past decade, major advances in imaging techniques have enhanced our understanding of Plasmodium spp. parasites and their interplay with mammalian hosts and mosquito vectors. Cryoelectron tomography, cryo-X-ray tomography and super-resolution microscopy have shifted paradigms of sporozoite and gametocyte structure, the process of erythrocyte invasion by merozoites, and the architecture of Maurer's clefts. Intravital time-lapse imaging has been revolutionary for our understanding of pre-erythrocytic stages of rodent Plasmodium parasites. Furthermore, high-speed imaging has revealed the link between sporozoite structure and motility, and improvements in time-lapse microscopy have enabled imaging of the entire Plasmodium falciparum erythrocytic cycle and the complete Plasmodium berghei pre-erythrocytic stages for the first time. In this Review, we discuss the contribution of key imaging tools to these and other discoveries in the malaria field over the past 10 years.

The invasive form of the apicomplexan parasite Babesia divergens, the free merozoite, invades the erythrocytes of host vertebrates, leading to significant pathology. Although invasion is an active process critical for parasite survival, it is not yet entirely understood. Using techniques to isolate the viable free merozoite, as well as electron microscopy, we undertook a detailed morphological study and explored the sub-cellular structure of the invasive B. divergens free merozoite after it had left the host cell. We examined characteristic apicomplexan features such as the apicoplast, the inner and discontinuous double membrane complex, and the apical complex; some aspects of erythrocyte entry by B. divergens were also defined by electron microscopy. This study adds to our understanding of B. divergens free merozoites and their invasion of human erythrocytes., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The genome sequences of Eimeria tenella have been sequenced, but >70% of these genes are currently categorized as having an unknown function or annotated as conserved hypothetical proteins, and few of them have been studied. In the present study, a conserved hypothetical protein gene of E. tenella, designated EtCHP559, was cloned using rapid amplification of cDNA 5'-ends (5'RACE) based on the expressed sequence tag (EST). The 1746-bp full-length cDNA of EtCHP559 contained a 1224-bp open reading frame (ORF) that encoded a 407-amino acid polypeptide with the predicted molecular weight of 46.04 kDa. Real-time quantitative PCR analysis revealed that EtCHP559 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second generation merozoites). The ORF was inserted into pCold-TF to produce recombinant EtCHP559. Using western blotting, the recombinant protein was successfully recognized by rabbit serum against E. tenella sporozoites. Immunolocalization by using EtCHP559 antibody showed that EtCHP559 was mainly distributed on the parasite surface in free sporozoites and became concentrated in the anterior region after sporozoites were incubated in complete medium. The EtCHP559 became uniformly dispersed in immature and mature schizonts. Inhibition of EtCHP559 function using anti-rEtCHP559 polyclonal antibody reduced the ability of E. tenella sporozoites to invade host cells by >70%. Animal challenge experiments demonstrated that the recombinant EtCHP559 significantly increased the average body weight gain, reduced the oocyst outputs, alleviated cecal lesions of the infected chickens, and resulted in anticoccidial index >160 against E. tenella. These results suggest that EtCHP559 plays an important role in sporozoite invasion and could be an effective candidate for the development of a new vaccine against E. tenella.

Blood-stage replication of the human malaria parasite Plasmodium falciparum occurs via schizogony, wherein daughter parasites are formed by a specialized cytokinesis known as segmentation. Here we identify a parasite protein, which we name P. falciparum Merozoite Organizing Protein (PfMOP), as essential for cytokinesis of blood-stage parasites. We show that, following PfMOP knockdown, parasites undergo incomplete segmentation resulting in a residual agglomerate of partially divided cells. While organelles develop normally, the structural scaffold of daughter parasites, the inner membrane complex (IMC), fails to form in this agglomerate causing flawed segmentation. In PfMOP-deficient gametocytes, the IMC formation defect causes maturation arrest with aberrant morphology and death. Our results provide insight into the mechanisms of replication and maturation of malaria parasites.

To explore the primary stage or site of action of acetamizuril (AZL), a novel triazine anticoccidial compound, the ultrastructural development of Eimeria tenella at different endogenous stages was studied in experimentally infected chickens treated with a single oral dose of 15 mg/kg AZL. As a result of drug action, the differentiations of second-generation schizonts and microgamonts were largely inhibited and merozoites became irregular in shape. Meanwhile, the outer membrane blistering and perinuclear space enlargement were obvious in the second-generation schizonts and microgamonts, which were never observed in the classic triazine anticoccidiosis drug diclazuril-treated E. tenella. The chromatin aggregation, anachromasis, and marginalization were visible in merozoites and microgamonts. Furthermore, the abnormal evagination of microgametes finally resulted in the degeneration of microgamonts and the failure of subsequent fertilization. The most marked micromorphological alteration occurring in the macrogamonts was the WFB2. Even if the fertilization occurred, the formation of oocyst wall became malformed and the zygote proceeded to the obvious degeneration. In addition, mitochondria swelling and cytoplasm vacuolization were discerned in respective intracellular stages, while endoplasmic reticulum and Golgi body swelling was less seen. These alterations may be the causes leading to the final death of E. tenella and also provide some information for further exploring the mechanism of action of AZL at the molecular level.

Cystoisospora belli is an opportunistic protozoan that causes human cystoisosporiasis, an infection characterized by diarrhea, steatorrhea, abdominal pain, fever, and weight loss. The lack of animal models susceptible to C. belli, and the difficulty in obtaining clinical samples with fair amounts of oocysts have limited the research pertaining to the basic biology of this parasite. This study aimed to describe the ultrastructure of endogenous stages of C. belli in Monkey Rhesus Kidney Cells (MK2) and Human Ileocecal Adenocarcinoma cells (HCT-8). Zoites of C. belli exhibited typical morphological features of coccidia, which included a trilaminar pellicle, an apical complex formed by a conoid, polar rings, rhoptries, and micronemes, in addition to dense granules and the endoplasmic reticulum. No crystalloid body was observed but various lipid and amylopectin granules were usually present in the cytoplasm of zoites. We observed a tendency of the endoplasmic reticulum of the host cell to be located near the parasitophorous vacuole membrane. Merozoites were formed by endodyogeny and during replication, the apical complex of the mother cell remained intact. The formation of gametes or oocysts was not observed. The ultrastructural findings of C. belli are further evidence of its proximity to Sarcocystidae family members and corroborate their reclassification as Cystoisospora spp., (© 2014 Wiley Periodicals, Inc.)

Malaria remains a major health problem worldwide. All clinical symptoms of malaria are attributed to the asexual blood stages of the parasite life cycle. Proteins resident in apical organelles and present on the surface of P. falciparum merozoites are considered promising candidates for the development of blood stage malaria vaccines. In the present study, we have identified and characterized a microneme associated antigen, PfMA [PlasmoDB Gene ID: PF3D7_0316000, PFC0700c]. The gene was selected by applying a set of screening criteria such as transcriptional upregulation at late schizogony, inter-species conservation and the presence of signal sequence or transmembrane domains. The gene sequence of PfMA was found to be conserved amongst various Plasmodium species. We experimentally demonstrated that the transcript for PfMA was expressed only in the late blood stages of parasite consistent with a putative role in erythrocyte invasion. PfMA was localized by immunofluorescence and immuno-electron microscopy to be in the micronemes, an apical organelle of merozoites. The functional role of the PfMA protein in erythrocyte invasion was identified as a parasite adhesin involved in direct attachment with the target erythrocyte. PfMA was demonstrated to bind erythrocytes in a sialic acid independent, chymotrypsin and trypsin resistant manner and its antibodies inhibited P. falciparum erythrocyte invasion. Invasion of erythrocytes is a complex multistep process that involves a number of redundant ligand-receptor interactions many of which still remain unknown and even uncharacterized. Our work has identified and characterized a novel P. falciparum adhesin involved in erythrocyte invasion.

Erythrocyte invasion by merozoites forms of the malaria parasite is a key step in the establishment of human malaria disease. To date, efforts to understand cellular events underpinning entry have been limited to insights from non-human parasites, with no studies at sub-micrometer resolution undertaken using the most virulent human malaria parasite, Plasmodium falciparum. This leaves our understanding of the dynamics of merozoite sub-cellular compartments during infectionincomplete, in particular that of the secretory organelles. Using advances in P. falciparum merozoite isolation and new imaging techniques we present a three-dimensional study of invasion using electron microscopy, cryo-electron tomography and cryo-X-ray tomography. We describe the core architectural features of invasion and identify fusion between rhoptries at the commencement of invasion as a hitherto overlooked event that likely provides a critical step that initiates entry. Given the centrality of merozoite organelle proteins to vaccine development, these insights provide a mechanistic framework to understand therapeutic strategies targeted towards the cellular events of invasion., (© 2013 John Wiley & Sons Ltd.)

Background: The apicoplast is a plastid organelle derived from a secondary endosymbiosis, containing biosynthetic pathways essential for the survival of apicomplexan parasites. The Toxoplasma apicoplast clearly possesses four membranes but in related Plasmodium spp. the apicoplast has variably been reported to have either three or four membranes., Methods: Cryo-electron tomography was employed to image merozoites of Plasmodium falciparum and Plasmodium berghei frozen in their near-native state. Three-dimensional reconstructions revealed the number of apicoplast membranes and the association of the apicoplast with other organelles. Routine transmission electron microscopy of parasites preserved by high-pressure freezing followed by freeze substitution techniques was also used to analyse apicoplast morphology., Results: Cryo-preserved parasites showed clearly four membranes surrounding the apicoplast. A wider gap between the second and third apicoplast membranes was frequently observed. The apicoplast was found in close proximity to the nucleus and to the rhoptries. The apicoplast matrix showed ribosome-sized particles and membranous whorls., Conclusions: The Plasmodium apicoplast possesses four membranes, as do the apicoplasts of other apicomplexan parasites. This is consistent with a four-membraned secondary endosymbiotic plastid ancestor.

Export of proteins into the infected erythrocyte is critical for malaria parasite survival. The majority of effector proteins are thought to export via a proteinaceous translocon, resident in the parasitophorous vacuole membrane surrounding the parasite. Identification of the Plasmodium translocon of exported proteins and its biochemical association with exported proteins suggests it performs this role. Direct evidence for this, however, is lacking. Here using viable purified Plasmodium falciparum merozoites and three-dimensional structured illumination microscopy, we investigate remodelling events immediately following parasite invasion. We show that multiple complexes of the Plasmodium translocon of exported proteins localize together in foci that dynamically change in clustering behaviour. Furthermore, we provide conclusive evidence of spatial association between exported proteins and exported protein 2, a core component of the Plasmodium translocon of exported proteins, during native conditions and upon generation of translocation intermediates. These data provide the most direct cellular evidence to date that protein export occurs at regions of the parasitophorous vacuole membrane housing the Plasmodium translocon of exported proteins complex.

The Plasmodium falciparum histone deacetylase Sir2a localizes at telomeric regions where it contributes to epigenetic silencing of clonally variant virulence genes. Apart from telomeres, PfSir2a also accumulates in the nucleolus, which harbours the developmentally regulated ribosomal RNA genes. Here we investigate the nucleolar function of PfSir2a and demonstrate that PfSir2a fine-tunes ribosomal RNA gene transcription. Using a parasite line in which PfSir2a has been disrupted, we observe that histones near the transcription start sites of all ribosomal RNA genes are hyperacetylated and that transcription of ribosomal RNA genes is upregulated. Complementation of the PfSir2a-disrupted parasites restores the ribosomal RNA levels, whereas PfSir2a overexpression in wild-type parasites decreases ribosomal RNA synthesis. Furthermore, we observe that PfSir2a modulation of ribosomal RNA synthesis is linked to an altered number of daughter merozoites and the parasite multiplication rate. These findings provide new insights into an epigenetic mechanism that controls malaria parasite proliferation and virulence.

Plasmodium falciparum resistance to artemisinin derivatives, the first-line antimalarial drug, drives the search for new classes of chemotherapeutic agents. Current discovery is primarily directed against the intracellular forms of the parasite. However, late schizont-infected red blood cells (RBCs) may still rupture and cause disease by sequestration; consequently targeting invasion may reduce disease severity. Merozoite invasion of RBCs requires interaction between two parasite proteins AMA1 and RON2. Here we identify the first inhibitor of this interaction that also blocks merozoite invasion in genetically distinct parasites by screening a library of over 21,000 compounds. We demonstrate that this inhibition is mediated by the small molecule binding to AMA1 and blocking the formation of AMA1-RON complex. Electron microscopy confirms that the inhibitor prevents junction formation, a critical step in invasion that results from AMA1-RON2 binding. This study uncovers a strategy that will allow for highly effective combination therapies alongside existing antimalarial drugs.

The Apicomplexan parasite Cryptosporidium parvum is responsible for the widespread disease cryptosporidiosis, in both humans and livestock. The nature of C. parvum infection is far from understood and many questions remain in regard to host-parasite interactions, limiting successful treatment of the disease. To definitively identify a range of C. parvum stages in cell culture and to begin to investigate host cell interactions in some of the lesser known life stages, we have utilized a combined scanning electron microscopy and immunolabeling approach, correlating high resolution microstructural information with definitive immunogold labeling of Cryptosporidium stages. Several life cycle stages, including oocysts, merozoites I, trophozoites, gamonts and microgametocytes, were successfully immunolabeled in an in vitro model system. Developing oocysts were clearly immunolabeled, but this did not persist once excystation had occurred. Immunolabeling visualized on the host cell surface adjacent to invasive merozoites is likely to be indicative of receptor shedding, with merozoites also initiating host responses that manifested as abnormal microvilli on the host cell surface. Small sub-micron stages such as microgametocytes, which were impossible to identify as single entities without immunolabeling, were readily visualized and observed to attach to host cells via novel membranous projections. Epicellular parasites also expressed Cryptosporidium-derived epitopes within their encapsulating membrane. These data have allowed us to confidently identify a variety of C. parvum stages in cell culture at high resolution. With this, we provide new insight into C. parvum - host cell interactions and highlight future opportunities for investigating and targeting receptor-mediated interactions between Cryptosporidium life cycle stages and host cells., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The proteins P52 and P36 are expressed in the sporozoite stage of the murine malaria parasite Plasmodium berghei. Δp52&p36 sporozoites lacking expression of both proteins are severely compromised in their capability to develop into liver stage parasites and abort development soon after invasion; presumably due to the absence of a parasitophorous vacuole membrane (PVM). However, a small proportion of P. berghei Δp52&p36 parasites is capable to fully mature in hepatocytes causing breakthrough blood stage infections. We have studied the maturation of replicating Δp52&p36 parasites in cultured Huh-7 hepatocytes. Approximately 50% of Δp52&p36 parasites developed inside the nucleus of the hepatocyte but did not complete maturation and failed to produce merosomes. In contrast cytosolic Δp52&p36 parasites were able to fully mature and produced infectious merozoites. These Δp52&p36 parasites developed into mature schizonts in the absence of an apparent parasitophorous vacuole membrane as shown by immunofluorescence and electron microscopy. Merozoites derived from these maturing Δp52&p36 liver stages were infectious for C57BL/6 mice.

The protozoan parasite Plasmodium is transmitted by female Anopheles mosquitoes and undergoes obligatory development within a parasitophorous vacuole in hepatocytes before it is released into the bloodstream. The transition to the blood stage was previously shown to involve the packaging of exoerythrocytic merozoites into membrane-surrounded vesicles, called merosomes, which are delivered directly into liver sinusoids. However, it was unclear whether the membrane of these merosomes was derived from the parasite membrane, the parasitophorous vacuole membrane or the host cell membrane. This knowledge is required to determine how phagocytes will be directed against merosomes. Here, we fluorescently label the candidate membranes and use live cell imaging to show that the merosome membrane derives from the host cell membrane. We also demonstrate that proteins in the host cell membrane are lost during merozoite liberation from the parasitophorous vacuole. Immediately after the breakdown of the parasitophorous vacuole membrane, the host cell mitochondria begin to degenerate and protein biosynthesis arrests. The intact host cell plasma membrane surrounding merosomes allows Plasmodium to mask itself from the host immune system and bypass the numerous Kupffer cells on its way into the bloodstream. This represents an effective strategy for evading host defenses before establishing a blood stage infection.

The commitment of Plasmodium merozoites to invade red blood cells (RBCs) is marked by the formation of a junction between the merozoite and the RBC and the coordinated induction of the parasitophorous vacuole. Despite its importance, the molecular events underlying the parasite's commitment to invasion are not well understood. Here we show that the interaction of two parasite proteins, RON2 and AMA1, known to be critical for invasion, is essential to trigger junction formation. Using antibodies (Abs) that bind near the hydrophobic pocket of AMA1 and AMA1 mutated in the pocket, we identified RON2's binding site on AMA1. Abs specific for the AMA1 pocket blocked junction formation and the induction of the parasitophorous vacuole. We also identified the critical residues in the RON2 peptide (previously shown to bind AMA1) that are required for binding to the AMA1 pocket, namely, two conserved, disulfide-linked cysteines. The RON2 peptide blocked junction formation but, unlike the AMA1-specific Ab, did not block formation of the parasitophorous vacuole, indicating that formation of the junction and parasitophorous vacuole are molecularly distinct steps in the invasion process. Collectively, these results identify the binding of RON2 to the hydrophobic pocket of AMA1 as the step that commits Plasmodium merozoites to RBC invasion and point to RON2 as a potential vaccine candidate.

The most deadly of the human malaria parasites, Plasmodium falciparum, has different stages specialized for invasion of hepatocytes, erythrocytes, and the mosquito gut wall. In each case, host cell invasion is powered by an actin-myosin motor complex that is linked to an inner membrane complex (IMC) via a membrane anchor called the glideosome-associated protein 50 (PfGAP50). We generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 (PfGAP50-GFP). Using immunoprecipitation and fluorescence photobleaching, we show that C-terminally tagged PfGAP50-GFP can form a complex with endogenous copies of the linker protein PfGAP45 and the myosin A tail domain-interacting protein (MTIP). Full-length PfGAP50-GFP is located in the endoplasmic reticulum in early-stage parasites and then redistributes to apical caps during the formation of daughter merozoites. In the final stage of schizogony, the PfGAP50-GFP profile extends further around the merozoite surface. Three-dimensional (3D) structured illumination microscopy reveals the early-stage IMC as a doubly punctured flat ellipsoid that separates to form claw-shaped apposed structures. A GFP fusion of PfGAP50 lacking the C-terminal membrane anchor is misdirected to the parasitophorous vacuole. Replacement of the acid phosphatase homology domain of PfGAP50 with GFP appears to allow correct trafficking of the chimera but confers a growth disadvantage.

Erythrocyte invasion by the merozoite is an obligatory stage in Plasmodium parasite infection and essential to malaria disease progression. Attempts to study this process have been hindered by the poor invasion synchrony of merozoites from the only in vitro culture-adapted human malaria parasite, Plasmodium falciparum. Using fluorescence, three-dimensional structured illumination, and immunoelectron microscopy of filtered merozoites, we analyze cellular and molecular events underlying each discrete step of invasion. Monitoring the dynamics of these events revealed that commitment to the process is mediated through merozoite attachment to the erythrocyte, triggering all subsequent invasion events, which then proceed without obvious checkpoints. Instead, coordination of the invasion process involves formation of the merozoite-erythrocyte tight junction, which acts as a nexus for rhoptry secretion, surface-protein shedding, and actomyosin motor activation. The ability to break down each molecular step allows us to propose a comprehensive model for the molecular basis of parasite invasion., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Diclazuril, a benzeneacetonitrile anticoccidial agent, has potent activity against various stages of Eimeria tenella (E. tenella). To study the effects of diclazuril on E. tenella merozoites, purified second-generation merozoites were obtained from infected chicken caecal tissue at 120h after inoculation by a combination of enzymatic digestion, centrifugation, erythrocytes disruption and percoll density gradient centrifugation. Ultrastructural changes were monitored by transmission electron microscopy (TEM). Apoptosis and mitochondrial transmembrane potential were determined by flow cytometry (FCM). The results showed that diclazuril induced ultrastructural changes and significantly increased the ratio of early apoptosis by 180.75% (P<0.01) and late apoptosis/necrosis by 86.82% (P<0.05) in second-generation merozoites, respectively. Compared with the infected/control group, the ratio of second-generation merozoites that lost mitochondrial function was increased by 45.04% (P<0.01) in the infected/treatment group. In conclusion, diclazuril induced morphological changes and attenuated the activity of mitochondrial transmembrane potential of merozoites, which is involved in mitochondrial-depended apoptosis in second-generation merozoites of E. tenella.

The invasive blood stage of malaria parasites, merozoites, are complex entities specialized for the capture and entry of red blood cells. Their potential for vaccination and other anti-malaria strategies have attracted much research attention over the last 40 years, and there is now a considerable body of data relating to their biology. In this article some of the major advances over this period and remaining challenges are reviewed.

In the present study, sarcocysts of a Sarcocystis species infecting the gecko Hemidactylus turcicus was investigated by light and transmission electron microscopy. Six out of 26 (23%) H. turcicus were found to be infected with cysts of Sarcocystis. Examined muscle samples of different sites showed high intensity of infection in the tail and hind limb skeletal muscles. Microscopically visible cysts reached a mean size of 80 x 720 mum. These cysts are characterized by a light microscopically thick cyst wall and inner prominent septa dividing their interior into a large number of compartments enclosing the different zoites. Ultrastructural characteristics of the primary cyst wall and its long, mostly not upright protrusions were investigated. Two widely differentiated zoites (metrocytes and cyst merozoites) were clearly identified; they all showed the characteristic architecture of the Apicomplexa and especially that of the genus Sarcocystis. The about 5-7 mum sized cyst merozoites seemed to be differentiated into those being either poorly or richly supplied with reserve materials (amylopectin, lipids). This finding may indicate the existence of different developmental stages. Events of endodyogeny represented the only mode of reproduction inside the cysts. While comparing the morphology of these cysts with other descriptions of cysts in reptiles, they were described as a new species (Sarcocystis turcicii).

The biogenesis, organization and function of the rhoptries are not well understood. Antisera were prepared to synthetic peptides prepared as multiple antigenic peptides (MAPs) obtained from a Plasmodium yoelii merozoite rhoptry proteome analysis. The antisera were used in immunofluorescence and immunoelectron microscopy of schizont-infected erythrocytes. Twenty-seven novel rhoptry proteins representing proteases, metabolic enzymes, secreted proteins and hypothetical proteins, were identified in the body of the rhoptries by immunoelectron microscopy. The merozoite rhoptries contain a heterogeneous mixture of proteins that may initiate host cell invasion and establish intracellular parasite development.

The identification of proteins present on the surface of Plasmodium falciparum-infected red blood cells as well as of free merozoites has been widely considered as one of the main areas of research in the development of an antimalarial vaccine due to their involvement in the parasite's pathogenesis and invasion mechanisms. Major advances had been accomplished in this area thanks to the analysis of the reported genomic sequence of P. falciparum, allowing for the identification of genes encoding for putative integral membrane proteins. This study reports for the first time the transcription of the MAL8P1.3 gene, which codifies for a 25-kDa integral membrane protein of P. falciparum (FCB-2 strain), namely, Pf25-IMP. Western blot and immunofluorescence assays using goat polyclonal sera indicate that this protein is expressed in erythrocytic asexual blood stages. A highly robust, sensible, and specific receptor-ligand interaction assay allowed identification of two high activity binding peptides (HABPs) derived from Pf25-IMP: 30577 ((41)YKTANENVKLASSLSDRLSR(60)) and 30583 ((161)LNKKTVVRKIAEGLGYTIVF(180)). Both HABPs bound with high affinity to human red blood cells (RBCs), and such binding was susceptible to enzyme treatment with trypsin. A common RBC surface receptor of apparently 48 kDa was found for both HABPs, plus an additional 31-kDa receptor for HABP 30577. HABP 30577 inhibited merozoite invasion in vitro by 73%, while HABP 30583 showed a 59% inhibition at 200 microM concentration. The data suggest a possible role of Pf25-IMP in merozoite invasion to RBCs and support its inclusion in further immunological studies for evaluating its potential as vaccine candidates.

Morphological and genetic features of a new Hepatozoon species, Hepatozoon ursi n. sp., in Japanese black bear (Ursus thibetanus japonicus) were studied. Schizogonic developmental stages were observed in the lungs of Japanese black bears. The schizonts were sub-spherical in shape and 45.7+/-4.6 x 42.7+/-4.5 microm in size. Each mature schizont contained approximately 80-130 merozoites and 0-5 residual bodies. The merozoites were 7.0+/-0.7 x 1.8+/-0.3 microm in size. Intraleukocytic gametocytes were slightly curved, cigar-like in shape and had a beak-like protrusion at one end. The size of the gametocytes was 10.9+/-0.3 x 3.3+/-0.2 microm. The analyses of the18S rRNA gene sequences supported the hypothesis that H. ursi n. sp. is different from other Hepatozoon species. Mature Hepatozoon oocysts were detected in two species of ticks (Haemaphysalis japonica and Haemaphysalis flava) collected on the bears infected with H. ursi n. sp. Two measured oocysts were 263.2 x 234.0 microm and 331.8 x 231.7 microm, respectively. The oocysts contained approximately 40 and 50 sporocysts, respectively. The sporocysts were sub-spherical in shape and 31.2+/-2.5 x 27.0+/-2.9 microm in size. Each sporocyst contained at least 8-16 sporozoites, with the sporozoites being 12.2+/-1.4 x 3.5+/-0.5 microm in size. H. ursi n. sp. is the first Hepatozoon species recorded from the family Ursidae.

The life cycle of the apicomplexan protozoon Hepatozoon canis in its natural hosts Rhipicephalus sanguineus (tick) and Canis familiaris (domestic dog) was studied in an experimental infection. Tick nymphs were fed on a naturally infected dog, or they were infected by percutaneous injection of blood. Dogs were inoculated by ingestion of adult ticks containing mature oocysts. Gamonts were in syzygy 24 hr after percutaneous injection of ticks. Early oocysts were detected 96 hr after nymph repletion, and mature oocysts in adult ticks were infective to dogs 40 days postmolt. Merogony was detected in dog bone marrow from 13 days postinoculation (PI) and included meronts containing 20-30 micromerozoites, and a second type with 2-4 macromerozoites. Monozoic cysts were observed in the spleen in conjunction with merogony. Gamontogony with infection of leukocytes by micromerozoites occurred from 26 days PI, and gamont parasitemia, which completed the life cycle, was detected 28 days PI. The length of the life cycle from nymphal attachment to parasitemia in dogs was 81 days. Increased body temperatures were evident from 16 to 27 days PI and paralleled the time of intensive bone marrow merogony. Skeletal pain and recumbency were manifested in 2 dogs. This study further elucidates the life cycle of H. canis and provides a sequential morphologic description of H. canis merogony, gamontogony, and sporogony.

Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter formation.

The ultrastructure of merozoites, gamonts and oocysts of the neogregarine Mattesia dispora and their development in larvae of the flour moth Ephestia kuehniella were studied by electron microscopy. The apical complex of free macronuclear merozoites was very distinct in micrographs of sections, the polar rings being especially prominent. Two gamonts associated in head-to-head syzygy and the apical complexes served as the contact point during pairing. At this stage the rhoptries became reduced and the conoid widened. The gamonts had a foam-like appearance in the light microscope. Paired gamonts formed an envelope and developed into a gametocyst, within which the gamonts were separated by a distinct border. Four gametes and two residual cells developed inside the gametocyst. The gametes were covered with a single membrane. The gametes fused in pairs to form two spherical zygotes, each covered by two membranes and with one large nucleus. The external layer appeared more undulated than the inner one. A single membrane covered each residual cell. Walls were formed around both zygotes to produce two oocysts. Each mature oocyst was lemon-shaped with polar plugs and eight peripheral sporozoites, which had a pellicle similar to that of the merozoites, lay beneath the thick oocyst wall.

The present study focuses on and describes the developmental stages of a haemogregarine species in the blood and tissues of the gecko Ptyodactylus hasselquistii. The blood stages were differentiated into two forms: a short gamont measuring 11.87 x 6.42 microm and a banana-shaped mature gamont measuring 14.13 x 10.03 microm in length and width, respectively. Both erythrocytes and leucocytes had been invaded. The parasitaemia level is up to 410 per 10,000 erythrocytes counted. The gamont has a karyolytic effect on the host cell nucleus. Merogony occurred in the parenchyma cells of liver and the endothelial cells of the lung. The meronts in the lung were found in two forms: the micromeront measured 14.93x13.14 microm and produced a few numbers (average 4) of macromerozoites. The macromeront measured 26.3 x 16 microm and produced more small-sized merozoites (average 11.5), or micromerozoites. On the ultrastructural level, merozoites have a pellicle, which consists of an outer and inner membrane. The merozoites are nearly identical to the blood stages of the parasite.