An improved method for the in vitro differentiation of Plasmodium falciparum gametocytes into ookinetes

<p>Abstract</p> <p>Background</p> <p>Ookinete is the form of the malaria parasite that invades the mosquito midgut epithelium to initiate sporogony. Differentiation of ingested gametocytes into ookinetes in the mosquito midgut lumen and subsequent interaction with the lumenal surface of the midgut epithelium in preparation for invasion is a complex and multi-stepped process. To facilitate the study of these events in detail it is necessary to produce sufficient numbers of pure, fully mature and functional ookinetes. However, production of even a small number of <it>Plasmodium </it><it>falciparum </it>ookinetes <it>in vitro </it>has proven to be a daunting task. Consequently, over the past four decades our collective understanding of the biology of this parasite form remains sorely deficient. This article reports on investigations of five different ookinete media, in an effort to improve the <it>in vitro </it>transformation efficiency of <it>P. falciparum </it>gametocytes into mature ookinetes and their infectivity of the mosquito midgut.</p> <p>Methods</p> <p>Five different ookinete media were evaluated for their ability to support the differentiation of gametocytes into gametes and further into mature stage V ookinetes. Moreover, infectivity of the <it>in vitro</it>-transformed ookinetes was evaluated by feeding them to vector mosquitoes and measuring their ability to traverse the midgut and form oocysts.</p> <p>Results</p> <p>One of the five media (medium E) was clearly superior in that the cultured ookinetes produced the largest number of oocysts when fed to mosquitoes. Key components were additions of human serum, human red blood cell lysate and mosquito pupal extract, resulting in the production of larger numbers of ookinetes able to develop into oocysts when fed to mosquitoes.</p> <p>Conclusion</p> <p>This simple and practical improvement over the prevailing methodology will facilitate the investigation of how this important human malaria parasite initiates its development in the mosquito and will contribute to the understanding of its transmission biology.</p

Hemolytic C-Type Lectin CEL-III from Sea Cucumber Expressed in Transgenic Mosquitoes Impairs Malaria Parasite Development

The midgut environment of anopheline mosquitoes plays an important role in the development of the malaria parasite. Using genetic manipulation of anopheline mosquitoes to change the environment in the mosquito midgut may inhibit development of the malaria parasite, thus blocking malaria transmission. Here we generate transgenic Anopheles stephensi mosquitoes that express the C-type lectin CEL-III from the sea cucumber, Cucumaria echinata, in a midgut-specific manner. CEL-III has strong and rapid hemolytic activity toward human and rat erythrocytes in the presence of serum. Importantly, CEL-III binds to ookinetes, leading to strong inhibition of ookinete formation in vitro with an IC50 of 15 nM. Thus, CEL-III exhibits not only hemolytic activity but also cytotoxicity toward ookinetes. In these transgenic mosquitoes, sporogonic development of Plasmodium berghei is severely impaired. Moderate, but significant inhibition was found against Plasmodium falciparum. To our knowledge, this is the first demonstration of stably engineered anophelines that affect the Plasmodium transmission dynamics of human malaria. Although our laboratory-based research does not have immediate applications to block natural malaria transmission, these findings have significant implications for the generation of refractory mosquitoes to all species of human Plasmodium and elucidation of mosquito–parasite interactions

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. © 2021, The Author(s).1

The glutathione biosynthetic pathway of Plasmodium is essential for mosquito transmission

1Infection of red blood cells (RBC) subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Plasmodium spp. have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. In this report, we analyzed a critical component of the GSH biosynthesis pathway using reverse genetics. Plasmodium berghei parasites lacking expression of gamma-glutamylcysteine synthetase (γ-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that γ-GCS is not essential for blood stage development. Despite a significant reduction in GSH levels, blood stage forms of pbggcs− parasites showed only a defect in growth as compared to wild type. In contrast, a dramatic effect on development of the parasites in the mosquito was observed. Infection of mosquitoes with pbggcs− parasites resulted in reduced numbers of stunted oocysts that did not produce sporozoites. These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito

Single-Cell transcriptomics to define Plasmodium falciparum stage transition in the mosquito midgut

Malaria inflicts the highest rate of morbidity and mortality among the vector-borne diseases. The dramatic bottleneck of parasite numbers that occurs in the gut of the obligatory mosquito vector provides a promising target for novel control strategies. Using single-cell transcriptomics, we analyzed Plasmodium falciparum development in the mosquito gut, from unfertilized female gametes through the first 20 h after blood feeding, including the zygote and ookinete stages. This study revealed the temporal gene expression of the ApiAP2 family of transcription factors and of parasite stress genes in response to the harsh environment of the mosquito midgut. Further, employing structural protein prediction analyses, we found several upregulated genes predicted to encode intrinsically disordered proteins (IDPs), a category of proteins known for their importance in regulation of transcription, translation, and protein-protein interactions. IDPs are known for their antigenic properties and may serve as suitable targets for antibody- or peptide-based transmission suppression strategies. In total, this study uncovers the P. falciparum transcriptome from early to late parasite development in the mosquito midgut, inside its natural vector, which provides an important resource for future malaria transmission-blocking initiatives. IMPORTANCE The malaria parasite Plasmodium falciparum causes more than half a million deaths per year. The current treatment regimen targets the symptom-causing blood stage inside the human host. However, recent incentives in the field call for novel interventions to block parasite transmission from humans to the mosquito vector. Therefore, we need to better understand the parasite biology during its development inside the mosquito, including a deeper understanding of the expression of genes controlling parasite progression during these stages. Here, we have generated single-cell transcriptome data, covering P. falciparum’s development, from gamete to ookinete inside the mosquito midgut, uncovering previously untapped parasite biology, including a repertoire of novel biomarkers to be explored in future transmission-blocking efforts. We anticipate that our study provides an important resource, which can be further explored to improve our understanding of the parasite biology as well as aid in guiding future malaria intervention strategies

Malaria Parasite Invasion of the Mosquito Salivary Gland Requires Interaction between the Plasmodium TRAP and the Anopheles Saglin Proteins

SM1 is a twelve-amino-acid peptide that binds tightly to the Anopheles salivary gland and inhibits its invasion by Plasmodium sporozoites. By use of UV-crosslinking experiments between the peptide and its salivary gland target protein, we have identified the Anopheles salivary protein, saglin, as the receptor for SM1. Furthermore, by use of an anti-SM1 antibody, we have determined that the peptide is a mimotope of the Plasmodium sporozoite Thrombospondin Related Anonymous Protein (TRAP). TRAP binds to saglin with high specificity. Point mutations in TRAP's binding domain A abrogate binding, and binding is competed for by the SM1 peptide. Importantly, in vivo down-regulation of saglin expression results in strong inhibition of salivary gland invasion. Together, the results suggest that saglin/TRAP interaction is crucial for salivary gland invasion by Plasmodium sporozoites

Interrupting malaria transmission by genetic manipulation of anopheline mosquitoes

Malaria ranks among the deadliest infectious diseases that kills more than one million persons everyyear. The mosquito is an obligatory vector for malaria transmission. In the mosquito, Plasmodiumundergoes a complex series of developmental events that includes transformation into severaldistinct morphological forms and the crossing of two different epithelia—midgut and salivarygland. Circumstantial evidence suggests that crossing of the epithelia requires specific interactionsbetween Plasmodium and epithelial surface molecules. By use of a phage display library we haveidentified a small peptide-SM1—that binds to the surfaces of the mosquito midgut and salivaryglands. Transgenic Anopheles stephensi mosquitoes expressing a SM1 tetramer from a bloodinducibleand gut-specific promoter are substantially impaired in their ability to sustain parasitedevelopment and transmission. A second effector gene, phospholipase A2, also impairs parasitetransmission in transgenic mosquitoes. These findings have important implications for the developmentof new strategies for malaria control