Correlative light-electron microscopy methods to characterize the ultrastructural features of the replicative and dormant liver stages of Plasmodium parasites.

BACKGROUND: The infection of the liver by Plasmodium parasites is an obligatory step leading to malaria disease. Following hepatocyte invasion, parasites differentiate into replicative liver stage schizonts and, in the case of Plasmodium species causing relapsing malaria, into hypnozoites that can lie dormant for extended periods of time before activating. The liver stages of Plasmodium remain elusive because of technical challenges, including low infection rate. This has been hindering experimentations with well-established technologies, such as electron microscopy. A deeper understanding of hypnozoite biology could prove essential in the development of radical cure therapeutics against malaria. RESULTS: The liver stages of the rodent parasite Plasmodium berghei, causing non-relapsing malaria, and the simian parasite Plasmodium cynomolgi, causing relapsing malaria, were characterized in human Huh7 cells or primary non-human primate hepatocytes using Correlative Light-Electron Microscopy (CLEM). Specifically, CLEM approaches that rely on GFP-expressing parasites (GFP-CLEM) or on an immunofluorescence assay (IFA-CLEM) were used for imaging liver stages. The results from P. berghei showed that host and parasite organelles can be identified and imaged at high resolution using both CLEM approaches. While IFA-CLEM was associated with more pronounced extraction of cellular content, samples features were generally well preserved. Using IFA-CLEM, a collection of micrographs was acquired for P. cynomolgi liver stage schizonts and hypnozoites, demonstrating the potential of this approach for characterizing the liver stages of Plasmodium species causing relapsing malaria. CONCLUSIONS: A CLEM approach that does not rely on parasites expressing genetically encoded tags was developed, therefore suitable for imaging the liver stages of Plasmodium species that lack established protocols to perform genetic engineering. This study also provides a dataset that characterizes the ultrastructural features of liver stage schizonts and hypnozoites from the simian parasite species P. cynomolgi

Correlative light-electron microscopy methods to characterize the ultrastructural features of the replicative and dormant liver stages of Plasmodium parasites

Abstract Background The infection of the liver by Plasmodium parasites is an obligatory step leading to malaria disease. Following hepatocyte invasion, parasites differentiate into replicative liver stage schizonts and, in the case of Plasmodium species causing relapsing malaria, into hypnozoites that can lie dormant for extended periods of time before activating. The liver stages of Plasmodium remain elusive because of technical challenges, including low infection rate. This has been hindering experimentations with well-established technologies, such as electron microscopy. A deeper understanding of hypnozoite biology could prove essential in the development of radical cure therapeutics against malaria. Results The liver stages of the rodent parasite Plasmodium berghei, causing non-relapsing malaria, and the simian parasite Plasmodium cynomolgi, causing relapsing malaria, were characterized in human Huh7 cells or primary non-human primate hepatocytes using Correlative Light-Electron Microscopy (CLEM). Specifically, CLEM approaches that rely on GFP-expressing parasites (GFP-CLEM) or on an immunofluorescence assay (IFA-CLEM) were used for imaging liver stages. The results from P. berghei showed that host and parasite organelles can be identified and imaged at high resolution using both CLEM approaches. While IFA-CLEM was associated with more pronounced extraction of cellular content, samples’ features were generally well preserved. Using IFA-CLEM, a collection of micrographs was acquired for P. cynomolgi liver stage schizonts and hypnozoites, demonstrating the potential of this approach for characterizing the liver stages of Plasmodium species causing relapsing malaria. Conclusions A CLEM approach that does not rely on parasites expressing genetically encoded tags was developed, therefore suitable for imaging the liver stages of Plasmodium species that lack established protocols to perform genetic engineering. This study also provides a dataset that characterizes the ultrastructural features of liver stage schizonts and hypnozoites from the simian parasite species P. cynomolgi. Graphical Abstrac

A rapid and scalable density gradient purification method for <it>Plasmodium</it> sporozoites

Abstract Background Malaria remains a major human health problem, with no licensed vaccine currently available. Malaria infections initiate when infectious Plasmodium sporozoites are transmitted by Anopheline mosquitoes during their blood meal. Investigations of the malaria sporozoite are, therefore, of clear medical importance. However, sporozoites can only be produced in and isolated from mosquitoes, and their isolation results in large amounts of accompanying mosquito debris and contaminating microbes. Methods Here is described a discontinuous density gradient purification method for Plasmodium sporozoites that maintains parasite infectivity in vitro and in vivo and greatly reduces mosquito and microbial contaminants. Results This method provides clear advantages over previous approaches: it is rapid, requires no serum components, and can be scaled to purify >107 sporozoites with minimal operator involvement. Moreover, it can be effectively applied to both human (Plasmodium falciparum, Plasmodium vivax) and rodent (Plasmodium yoelii) infective species with excellent recovery rates. Conclusions This novel method effectively purifies viable malaria sporozoites by greatly reducing contaminating mosquito debris and microbial burdens associated with parasite isolation. Large-scale preparations of purified sporozoites will allow for enhanced in vitro infections, proteomics, and biochemical characterizations. In conjunction with aseptic mosquito rearing techniques, this purification technique will also support production of live attenuated sporozoites for vaccination.</p

PfCap380 as a marker for Plasmodium falciparum oocyst development in vivo and in vitro

Abstract Background Despite the importance of the Plasmodium berghei oocyst capsule protein (PbCap380) in parasite survival, very little is known about the orthologous Plasmodium falciparum capsule protein (PfCap380). The goal of this work was to study the growth of P. falciparum oocysts using PfCap380 as a developmental marker. Methods To study P. falciparum oocyst development using both in vivo (mosquito-derived) and in vitro (culture-derived) growth conditions, antibodies (polyclonal antisera) were raised against PfCap380. For studies on in vivo oocysts, mature P. falciparum gametocytes were fed to Anopheles stephensi mosquitoes. For studies on in vitro parasites, P. falciparum gametocytes were induced and matured for subsequent ookinete production. Ookinetes were purified and then tested for binding affinity to basal lamina components and transformation into early oocysts, which were grown on reconstituted basal lamia coated wells with novel oocyst media. To monitor in vivo oocyst development, immunofluorescence assays (IFA) were performed using anti-PfCap380 antisera on Pf-infected mosquito midguts. IFA were also performed on culture-derived oocysts to follow in vitro oocyst development. Results The anti-PfCap380 antisera allowed detection of early midgut oocysts starting at 2 days after gametocyte infection, while circumsporozoite protein was definitively observed on day 6. For in vitro culture, significant transformation of gametocytes to ookinetes (24%) and of ookinetes to early oocysts (85%) was observed. After screening several basal lamina components, collagen IV provided greatest binding of ookinetes and transformation into early oocysts. Finally, PfCap380 expression was observed on the surface of culture-derived oocysts but not on gametocytes or ookinetes. Conclusions This study presents developmental monitoring of P. falciparum oocysts produced in vivo and in vitro. The anti-PfCap380 antisera serves as an important reagent for developmental studies of oocysts from the mosquito midgut and also from oocyst culture using in vitro methodology. The present data demonstrate that PfCap380 is a useful marker to follow the development and maturation of in vivo and in vitro produced oocysts as early as 2 days after zygote formation. Further in vitro studies focused on oocyst and sporozoite maturation will support the manufacturing of whole sporozoites for malaria vaccines

MOESM5 of PfCap380 as a marker for Plasmodium falciparum oocyst development in vivo and in vitro

Additional file 5: Figure S5. Ookinetes do not express PfCap380. IFA were performed using purified ookinetes with anti-PfCap380 antisera directly labeled with Alexa Fluor 594. Ookinetes (1-4) express GFP in green and nuclei stain with DAPI in blue but do not express PfCap380 (red). The merged image of the four separate channels is shown. Scale bar = 5 Îźm

MOESM4 of PfCap380 as a marker for Plasmodium falciparum oocyst development in vivo and in vitro

Additional file 4: Figure S4. Transformation rates between parasite stages. The graph shows the transformation rates for gametocyte to ookinete stages and ookinete to early oocyst stages. The gametocyte to ookinete transformation rate was determined by counting gametocytes and ookinetes in a hemocytometer. The ookinete to oocyst transformation rate was determined by counting oocysts in an 8-well-chamber slide that formed after seeding a known quantity of ookinetes. The values depict averages across three experiments and error bars represent standard deviation

MOESM3 of PfCap380 as a marker for Plasmodium falciparum oocyst development in vivo and in vitro

Additional file 3: Figure S3. Negative control IFA on in vivo and in vitro oocysts. IFA were performed as described except no primary antisera was used to test secondary antibodies for non-specific binding. Secondary antibodies were used to label midgut oocysts (A) or in vitro oocysts (B) and show expression of GFP in green and DAPI nuclear staining in blue. The merged image of the three separate channels is shown. DIC images were taken for in vitro but not in vivo oocysts due to challenges in imaging midgut tissue. Scale bars = 10 Îźm. For midgut oocysts, negative control antibodies used were Alexa Fluor 594 anti-rabbit for Cap380 and Alexa Fluor 647 anti-mouse for CSP. For in vitro oocysts, negative control antibodies used were Alexa Fluor 594 anti-rabbit for Cap380 and Alexa Fluor 594 anti-mouse for CSP

MOESM2 of PfCap380 as a marker for Plasmodium falciparum oocyst development in vivo and in vitro

Additional file 2: Figure S2. Expression of the PfCap380 peptide antigen. An image of an SDS-PAGE protein gel shows the migration of the purified His-tagged-PfCap380 peptide band in A (arrow). Western blot analysis shows the same peptide fragment recognized by an anti-His antibody in panel B (arrow). Protein molecular weight markers (10-120 or 22-120 kDa) are indicated. These results were generated by GenScript and shown with their permission

MOESM1 of PfCap380 as a marker for Plasmodium falciparum oocyst development in vivo and in vitro

Additional file 1: Figure S1. Sequence alignment of Plasmodium Cap380 orthologues. Amino acid sequence alignment of the PfCap380 proteins for several Plasmodium species was performed to identify similarities within the peptide immunogen region. In A, Asterisks = fully conserved residues, colon = conservation with strongly similar properties, period = conservation with weakly similar properties. In B, the percentages of similar, identical, or gap amino acids are shown for the PfCap380 antigen compared to Plasmodium species. PfCap380 shares most amino acid similarity with Pg, followed by Pv, Pb, and then Py