Reality check for malaria proteomics

Post-translationally modified protein isoforms are common in red blood cell stages of the malaria parasite

A malaria membrane skeletal protein is essential for normal morphogenesis, motility, and infectivity of sporozoites

Membrane skeletons are structural elements that provide mechanical support to the plasma membrane and define cell shape. Here, we identify and characterize a putative protein component of the membrane skeleton of the malaria parasite. The protein, named PbIMC1a, is the structural orthologue of the Toxoplasma gondii inner membrane complex protein 1 (TgIMC1), a component of the membrane skeleton in tachyzoites. Using targeted gene disruption in the rodent malaria species Plasmodium berghei, we show that PbIMC1a is involved in sporozoite development, is necessary for providing normal sporozoite cell shape and mechanical stability, and is essential for sporozoite infectivity in insect and vertebrate hosts. Knockout of PbIMC1a protein expression reduces, but does not abolish, sporozoite gliding locomotion. We identify a family of proteins related to PbIMC1a in Plasmodium and other apicomplexan parasites. These results provide new functional insight in the role of membrane skeletons in apicomplexan parasite biology

A Plasmodium falciparum Strain Expressing GFP throughout the Parasite's Life-Cycle

The human malaria parasite Plasmodium falciparum is responsible for the majority of malaria-related deaths. Tools allowing the study of the basic biology of P. falciparum throughout the life cycle are critical to the development of new strategies to target the parasite within both human and mosquito hosts. We here present 3D7HT-GFP, a strain of P. falciparum constitutively expressing the Green Fluorescent Protein (GFP) throughout the life cycle, which has retained its capacity to complete sporogonic development. The GFP expressing cassette was inserted in the Pf47 locus. Using this transgenic strain, parasite tracking and population dynamics studies in mosquito stages and exo-erythrocytic schizogony is greatly facilitated. The development of 3D7HT-GFP will permit a deeper understanding of the biology of parasite-host vector interactions, and facilitate the development of high-throughput malaria transmission assays and thus aid development of new intervention strategies against both parasite and mosquito

Plasmodium berghei calcium-dependent protein kinase 3 is required for ookinete gliding motility and mosquito midgut invasion

Apicomplexan parasites critically depend on a unique form of gliding motility to colonize their hosts and to invade cells. Gliding requires different stage and species-specific transmembrane adhesins, which interact with an intracellular motor complex shared across parasite stages and species. How gliding is regulated by extracellular factors and intracellular signalling mechanisms is largely unknown, but current evidence suggests an important role for cytosolic calcium as a second messenger. Studying a Plasmodium berghei gene deletion mutant, we here provide evidence that a calcium-dependent protein kinase, CDPK3, has an important function in regulating motility of the ookinete in the mosquito midgut. We show that a cdpk3(–) parasite clone produces morphologically normal ookinetes, which fail to engage the midgut epithelium, due to a marked reduction in their ability to glide productively, resulting in marked reduction in malaria transmission to the mosquito. The mutant was successfully complemented with an episomally maintained cdpk3 gene, restoring mosquito transmission to wild-type level. cdpk3(–) ookinetes maintain their full genetic differentiation potential when microinjected into the mosquito haemocoel and cdpk3(–) sporozoites produced in this way are motile and infectious, suggesting an ookinete-limited essential function for CDPK3

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

A semi-automated method for counting fluorescent malaria oocysts increases the throughput of transmission blocking studies

<p>Abstract</p> <p>Background</p> <p>Malaria transmission is now recognized as a key target for intervention. Evaluation of the <it>Plasmodium </it>oocyst burden in the midguts of <it>Anopheles spp</it>. is important for many of assays investigating transmission. However, current assays are very time-consuming, manually demanding and patently subject to observer-observer variation.</p> <p>Methods</p> <p>This report presents the development of a method to rapidly, accurately and consistently determine oocyst burdens on mosquito midguts using GFP-expressing <it>Plasmodium berghei </it>and a custom-written macro for ImageJ. The counting macro was optimized and found to be fit-for-purpose by performing gametocyte membrane feeds with parasite infected blood. Dissected midguts were counted both manually and using the automated macro, then compared. The optimized settings for the macro were then validated by using it to determine the transmission blocking efficacies of two anti-malarial compounds - dehydroepiandrosterone sulphate and lumefantrine, in comparison to manually determined analysis of the same experiment.</p> <p>Results</p> <p>Concurrence of manual and macro counts was very high (R²= 0.973) and reproducible. Estimated transmission blocking efficacies between manual and automated analysis were highly concordant, indicating that dehydroepiandrosterone sulphate has little or no transmission blocking potential, whilst lumefantrine strongly inhibits sporogony.</p> <p>Conclusion</p> <p>Recognizing a potential five-fold increase in throughput, the resulting reduction in personnel costs, and the absence of inter-operator/laboratory variation possible with this approach, this counting macro may be a benefit to the malaria community.</p

Debris-Collecting Vacuum Machine with Grounded Safety System and Associated Methods

A debris collection machine includes a vacuum system (including a suction source operable to provide suction for pulling debris into a receptacle), a ground reference portion, a ground test portion, and a ground-checking module. The ground reference portion is electrically coupled with an electrically grounded reference point, and the ground test portion is electrically coupled with a portion of the vacuum system. The ground-checking module determines a resistance between from the ground reference portion and the ground test portion and prevents or terminates operation of the suction source of the vacuum system when the resistance exceeds a predetermined threshold value, e.g., which may correspond to a risk condition of spark generation that could ignite material in the receptacle

The Armadillo Repeat Protein PF16 Is Essential for Flagellar Structure and Function in Plasmodium Male Gametes

Malaria, caused by the apicomplexan parasite Plasmodium, threatens 40% of the world\u27s population. Transmission between vertebrate and insect hosts depends on the sexual stages of the life-cycle. The male gamete of Plasmodium parasite is the only developmental stage that possesses a flagellum. Very little is known about the identity or function of proteins in the parasite\u27s flagellar biology. Here, we characterise a Plasmodium PF16 homologue using reverse genetics in the mouse malaria parasite Plasmodium berghei. PF16 is a conserved Armadillo-repeat protein that regulates flagellar structure and motility in organisms as diverse as green algae and mice. We show that P. berghei PF16 is expressed in the male gamete flagellum, where it plays a crucial role maintaining the correct microtubule structure in the central apparatus of the axoneme as studied by electron microscopy. Disruption of the PF16 gene results in abnormal flagellar movement and reduced fertility, but does not lead to complete sterility, unlike pf16 mutations in other organisms. Using homology modelling, bioinformatics analysis and complementation studies in Chlamydomonas, we show that some regions of the PF16 protein are highly conserved across all eukaryotes, whereas other regions may have species-specific functions. PF16 is the first ARM-repeat protein characterised in the malaria parasite genus Plasmodium and this study opens up a novel model for analysis of Plasmodium flagellar biology that may provide unique insights into an ancient organelle and suggest novel intervention strategies to control the malaria parasite