Anti-PfGARP activates programmed cell death of parasites and reduces severe malaria.

Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children1, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites2, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.

The Trypanosoma brucei subpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins.

Microtubules are inherently dynamic cytoskeletal polymers whose length and organization can be altered to perform essential functions in eukaryotic cells, such as providing tracks for intracellular trafficking and forming the mitotic spindle. Microtubules can be bundled to create more stable structures that collectively propagate force, such as in the flagellar axoneme, which provides motility. The subpellicular microtubule array of the protist parasite Trypanosoma brucei, the causative agent of African sleeping sickness, is a remarkable example of a highly specialized microtubule bundle. It is comprised of a single layer of microtubules that are crosslinked to each other and to the overlying plasma membrane. The array microtubules appear to be highly stable and remain intact throughout the cell cycle, but very little is known about the pathways that tune microtubule properties in trypanosomatids. Here, we show that the subpellicular microtubule array is organized into subdomains that consist of differentially localized array-associated proteins at the array posterior, middle, and anterior. The array-associated protein PAVE1 stabilizes array microtubules at the cell posterior and is essential for maintaining its tapered shape. PAVE1 and the newly identified protein PAVE2 form a complex that binds directly to the microtubule lattice, demonstrating that they are a true kinetoplastid-specific MAP. TbAIR9, which localizes to the entirety of the subpellicular array, is necessary for maintaining the localization of array-associated proteins within their respective subdomains of the array. The arrangement of proteins within the array likely tunes the local properties of array microtubules and creates the asymmetric shape of the cell, which is essential for parasite viability.

Single cell transcriptional changes across the blood stages of artemisinin resistant K13C580Y mutant Plasmodium falciparum upon dihydroartemisinin exposure.

Artemisinins have been a cornerstone of malaria control, but resistance in Plasmodium falciparum, due to mutations in the Kelch 13 gene, threaten these advances. Artemisinin exposure results in a dynamic transcriptional response across multiple pathways, but most work has focused on ring stages and ex vivo transcriptional analysis, limiting evaluation of all life cycle stages. We applied single cell RNAseq to two unsynchronized isogenic parasite lines (K13C580 and K13580Y) over 6 hrs after a pulse exposure to dihydroartemisinin (DHA). Transcription was altered across all stages, with the greatest occurring at the early trophozoite and mid ring stage in both lines. This response involved the arrest of metabolic processes and the enhancement of protein trafficking and the unfolded protein response. While similar, the response was enhanced in the K13580Y mutant, which may lead to the dormancy phenomenon upon treatment. Increased surface protein expression was seen in mutant parasites at baseline and upon drug exposure, highlighted by the increased expression of PfEMP1 and GARP, a potential therapeutic target. Antibody targeting GARP maintained anti-parasitic efficacy in mutant parasites. This work provides single cell insight of gene transcription across all life cycle stages revealing transcriptional changes that could initiate dormancy state and mediate survival.

Protocol for Differential Biopanning of P. falciparum Phage Display cDNA Library to Identify Parasite Targets of Protective Antibodies.

Malaria remains a significant global health burden, killing hundreds of thousands of children annually (WHO, The world malaria report. WHO, Geneva, 2019). Despite decades of effort, no broadly effective vaccine exists. Differential screening of parasite phage display libraries is a promising approach to identify the targets of human antibodies expressed by resistant but not by susceptible individuals (Raj et al., Nature, 582, 104-108, 2020; Science, 344, 871-877, 2014). Our whole proteome differential screening (WPDS) approach consists of positive selection to capture phage that bind antibodies expressed by malaria-resistant individuals, followed by negative selection to remove phage that bind antibodies expressed by malaria-susceptible individuals, and amplification of differentially recognized clones.

Assessing PfGARP-Mediated Apoptosis of Blood-Stage Plasmodium falciparum Parasites.

Apoptosis is conventionally regarded as an evolutionarily conserved and genetically controlled process of programmed cell death confined to metazoan organisms. However, recently, conserved features of apoptosis have also been demonstrated in unicellular eukaryotes (Holzmuller et al. Parasitology 132:S19-S32, 2006; Le Chat et al. Mol Biochem Parasitol 153:41-47, 2007; Madeo et al. Curr Opin Microbiol 7:655-660, 2004; Welburn et al. Parasitology 132:S7-S18, 2006; Jensen et al. Science 216:1230-1233, 1982) including malaria parasites (Al-Olayan et al. Int J Parasitol 32:1133-1143, 2002; Ch'ng et al. Cell Death Dis 1:e26, 2010; Meslin et al. J Infect Dis 195:1852-1859, 2007; Picot et al. Trans R Soc Trop Med Hyg 91:590-591, 1997; Raj et al. Nature 582:104-108, 2020). P. falciparum glutamic-acid-rich protein (PfGARP) is an antigen of 80 kDa that is uniquely expressed on the exofacial surface of red blood cells (RBCs) infected by early-to-late-trophozoite-stage P. falciparum parasites (Raj et al. Nature 582:104-108, 2020). We have recently demonstrated that antibodies against PfGARP bind to the PfGARP displayed on the surface of P. falciparum trophozoite-infected RBCs and trigger apoptosis in the intracellular parasites (Raj et al. Nature 582:104-108, 2020). This is the first demonstration of antibody-induced apoptosis in blood-stage malaria parasites and is characterized by several conserved features such as crisis form morphology, loss of mitochondrial membrane potential, loss of integrity of food vacuole, activation of caspase-like cysteine proteases, and fragmentation of chromosomal DNA. Here we describe the assays used to detect these features of apoptosis in the mature blood stage of malaria parasites.