Dissecting the biological content of host and parasite-derived extracellular vesicles in malaria

Plasmodium falciparum causes the deadliest form of malaria, especially in African children under five years. The parasite and its human host secrete small nanoparticles called extracellular vesicles (EVs) into their extracellular milieu. EVs contain bioactive molecules such as RNA and proteins reflecting that in the secreting cells. However, the biological contents of EVs are not well-characterized in the context of falciparum malaria. EVs are attractive sources of non-invasive biomarkers for diseases affecting inaccessible tissues such as the brain. Analysing the biological content of EVs will provide insights into their functions and could illuminate pathophysiological mechanisms of severe malaria syndromes, including cerebral malaria, respiratory distress, and severe malaria anaemia. The first part of this thesis aimed to investigate the biological benefit of EVs to the secreting parasite. To achieve this purpose, I sequenced four-hourly RNA samples obtained from EVs secreted by six P. falciparum isolates and compared them to the RNA within the secreting whole parasites. The data suggest parasite-derived EVs might be part of a post-transcriptional gene regulatory mechanism that maintains RNA homeostasis in P. falciparum. The second part of this thesis aimed to understand the pathophysiology of severe malaria complications. To this purpose, I quantified the protein and RNA content of plasma EVs obtained from acute malaria patients. I developed temporal models of disease progression by applying manifold learning to the cross-sectionally collected EV samples. In a parallel approach, I used plasma EV transcriptomes to develop a pseudo-temporal model of cerebral malaria. I determined that cerebral malaria might feature a progressive decrease in neural gene expression and an increase in glial gene expression, which can be followed using peripheral blood EVs. I conclude that EVs are treasure troves of biomarkers that can be used to eavesdrop on biological mechanisms in P. falciparum and its human host

The mRNA content of plasma extracellular vesicles provides a window into the brain during cerebral malaria disease progression

This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/The impact of cerebral malaria on the transcriptional profiles of cerebral tissue is difficult to study using non-invasive approaches. We isolated plasma extracellular vesicles (EVs) from patients with cerebral malaria and community controls and sequenced their RNA content. Deconvolution of the tissue origins of the EV-RNA revealed that EVs from cerebral malaria patients are predominantly enriched in transcripts of brain origin. Next, we used manifold learning on the EV-RNAseq data to determine pseudotime against the community control samples as the baseline reference. We found that neuronal transcripts in plasma EVs decreased as pseudotime progressed, while transcripts of glial, endothelial, and immune cell origins increased over pseudotime. Pseudotime was associated with clinicopathological parameters of disease severity, including retinopathy, metabolic acidosis, respiratory rate, anaemia, malnutrition, depth of unconsciousness and death. Plasma EVs further provided evidence of platelet activation, TNF signalling, neurotrophin signalling, long-term potentiation and glutamatergic signalling during late disease stages of cerebral malaria. The transcriptional responses of cerebral tissue in cerebral malaria can be studied non-invasively using EVs circulating in peripheral blood.Non peer reviewe

Novel stem cell technologies are powerful tools to understand the impact of human factors on Plasmodium falciparum malaria

© 2023 The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/Plasmodium falciparum parasites have a complex life cycle, but the most clinically relevant stage of the disease is the invasion of erythrocytes and the proliferation of the parasite in the blood. The influence of human genetic traits on malaria has been known for a long time, however understanding the role of the proteins involved is hampered by the a nuclear nature of erythrocytes that makes them inaccessible to genetic tools. Here we overcome this limitation using stem cells to generate erythroid cells with an in-vitro differentiation protocol and assess parasite invasion with an adaptation of flow cytometry to detect parasite hemozoin. We combine this strategy with reprogramming of patient cells to Induced Pluripotent Stem Cells and genome editing to understand the role of key genes and human traits in malaria infection. We show that deletion of basigin ablates invasion while deletion of ATP2B4 has a minor effect and that erythroid cells from reprogrammed patient-derived HbBart α-thalassemia samples poorly support infection. The possibility to obtain patient-secific and genetically modifed erythoid cells offers an unparalleled opportunity to study the role of human genes and polymorphisms in malaria allowing preservation of the genomic background to demonstrate their function and understand their mechanisms.Peer reviewe

Using Plasmodium knowlesi as a model for screening Plasmodium vivax blood-stage malaria vaccine targets reveals new candidates.

Plasmodium vivax is responsible for the majority of malaria cases outside Africa. Unlike P. falciparum, the P. vivax life-cycle includes a dormant liver stage, the hypnozoite, which can cause infection in the absence of mosquito transmission. An effective vaccine against P. vivax blood stages would limit symptoms and pathology from such recurrent infections, and therefore could play a critical role in the control of this species. Vaccine development in P. vivax, however, lags considerably behind P. falciparum, which has many identified targets with several having transitioned to Phase II testing. By contrast only one P. vivax blood-stage vaccine candidate based on the Duffy Binding Protein (PvDBP), has reached Phase Ia, in large part because the lack of a continuous in vitro culture system for P. vivax limits systematic screening of new candidates. We used the close phylogenetic relationship between P. vivax and P. knowlesi, for which an in vitro culture system in human erythrocytes exists, to test the scalability of systematic reverse vaccinology to identify and prioritise P. vivax blood-stage targets. A panel of P. vivax proteins predicted to function in erythrocyte invasion were expressed as full-length recombinant ectodomains in a mammalian expression system. Eight of these antigens were used to generate polyclonal antibodies, which were screened for their ability to recognize orthologous proteins in P. knowlesi. These antibodies were then tested for inhibition of growth and invasion of both wild type P. knowlesi and chimeric P. knowlesi lines modified using CRISPR/Cas9 to exchange P. knowlesi genes with their P. vivax orthologues. Candidates that induced antibodies that inhibited invasion to a similar level as PvDBP were identified, confirming the utility of P. knowlesi as a model for P. vivax vaccine development and prioritizing antigens for further follow up.European Union, National Institutes of Health (US

Extracellular vesicles could be a putative posttranscriptional regulatory mechanism that shapes intracellular RNA levels in Plasmodium falciparum

© 2023 The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/Plasmodium falciparum secretes extracellular vesicles (PfEVs) that contain parasite-derived RNA. However, the significance of the secreted RNA remains unexplored. Here, we compare secreted and intracellular RNA from asexual cultures of six P. falciparum lines. We find that secretion of RNA via extracellular vesicles is not only periodic throughout the asexual intraerythrocytic developmental cycle but is also highly conserved across P. falciparum isolates. We further demonstrate that the phases of RNA secreted via extracellular vesicles are discernibly shifted compared to those of the intracellular RNA within the secreting whole parasite. Finally, transcripts of genes with no known function during the asexual intraerythrocytic developmental cycle are enriched in PfEVs compared to the whole parasite. We conclude that the secretion of extracellular vesicles could be a putative posttranscriptional RNA regulation mechanism that is part of or synergise the classic RNA decay processes to maintain intracellular RNA levels in P. falciparum.Peer reviewe

Systemic and cerebrospinal fluid immune and complement activation in Ugandan children and adolescents with long‐standing nodding syndrome: a case‐control study

Objective Nodding syndrome is a poorly understood epileptic encephalopathy characterized by a unique seizure type— head nodding— and associated with Onchocerca volvulus infection. We hypothesized that altered immune activation in the cerebrospinal fluid (CSF) and plasma of children with nodding syndrome may yield insights into the pathophysiology and progression of this seizure disorder. Method We conducted a case‐control study of 154 children (8 years or older) with long‐standing nodding syndrome and 154 healthy age‐matched community controls in 3 districts of northern Uganda affected by nodding syndrome. Control CSF samples were obtained from Ugandan children in remission from haematological malignancy during routine follow‐up. Markers of immune activation and inflammation (cytokines and chemokines) and complement activation (C5a) were measured in plasma and CSF using ELISA or Multiplex Luminex assays. O. volvulus infection was assessed by serology for anti‐Ov16 IgG levels. Results The mean (SD) age of the population was 15.1 (SD 1.9) years and the mean duration of nodding syndrome from diagnosis to enrolment was 8.3 (SD 2.7) years. Majority with nodding syndrome had been exposed to O. volvulus 147/154 (95.4%) compared to community children 86/154 (55.8%), OR 17.04 (95% CI 7.33, 45.58), p<0.001. C5a was elevated in CSF of children with nodding syndrome compared to controls, (p<0.0001). The levels of other CSF markers tested were comparable between cases and controls after adjusting for multiple comparisons. Children with nodding syndrome had lower plasma levels of IL10, APRIL, CCL5 (RANTES), CCL2, CXCL13, MMP‐9 compared to community controls (p<0.05 for all; multiple comparisons). Plasma CRP was elevated in children with nodding syndrome compared to community children and correlated with disease severity. Significance Nodding syndrome is associated with exposure to O. volvulus. Compared to controls, children with long‐standing symptoms of nodding syndrome show evidence of complement activation in CSF and altered immune activation in plasma

Transnational connections of first generation immigrants from Kenya in the United States

This study analyzed why and how first generation immigrants from Kenya maintain transnational ties. It explored the characteristics of these ties, how they are sustained, whether they vary by gender, age, education or length of stay, and how ties affect immigrants' experiences. Ethnographic interviews with 38 participants living in Paterson, NJ showed no overarching immigrant experience. All participants regardless of age, gender, length of stay and education maintained transnational ties with family and friends. Ties took the form of phone calls, internet communication, mail, material exchanges, home visits and cultural activities and occurred mostly with people from their local ethnic villages. Frequency of ties by length of stay assumed a U-shaped curve with more ties initially, followed by a decline after some years in the U.S. and an increase thereafter. Participants mentioned three factors necessary for successful immigration experience: legal status in the U.S., a good education and a strong support network. None of them believed aspiring to a middle class American life or assimilation indicated success. Rather success meant assisting people in Kenya and co-ethnics validated the importance of transnational practices. Despite the absence of a visible Kenyan ethnic enclave in Paterson, there was a close-knit community connected through social networks. Women received assistance from kin and non-kin to migrate, while men were assisted mostly by family. Women's friendship ties transcended family ties but were not in competition with them; they used their friendship ties to advance family livelihoods. Men's immigration experiences were confounded by gender expectations based on their responsibilities as breadwinners and heads of households. Women's immigration decisions were interwoven in their daily struggles to support their families. Immigration was a never-ending process, as women and men perpetuated the experience through assisting the immigration of their children and friends and helping newly arrived immigrants. Future research using a longitudinal approach would enable an understanding of successive immigrant generations and if they reproduced the patterns of their elders' transnational ties. Longitudinal study would also allow us to discern if the U-shaped pattern of ties found here represented a cross-sectional perspective or instead overtime ties ebbed and flowed.Ph.D.Includes abstractVitaIncludes bibliographical referencesby Maria Mwikali Kiok

Novel stem cell technologies are powerful tools to understand the impact of human factors on Plasmodium falciparum malaria

Plasmodium falciparum parasites have a complex life cycle, but the most clinically relevant stage of the disease is the invasion of erythrocytes and the proliferation of the parasite in the blood. The influence of human genetic traits on malaria has been known for a long time, however understanding the role of the proteins involved is hampered by the anuclear nature of erythrocytes that makes them inaccessible to genetic tools. Here we overcome this limitation using stem cells to generate erythroid cells with an in-vitro differentiation protocol and assess parasite invasion with an adaptation of flow cytometry to detect parasite hemozoin. We combine this strategy with reprogramming of patient cells to Induced Pluripotent Stem Cells and genome editing to understand the role of key genes and human traits in malaria infection. We show that deletion of basigin ablates invasion while deletion of ATP2B4 has a minor effect and that erythroid cells from reprogrammed patient-derived HbBart α-thalassemia samples poorly support infection. The possibility to obtain patient-secific and genetically modifed erythoid cells offers an unparalleled opportunity to study the role of human genes and polymorphisms in malaria allowing preservation of the genomic background to demonstrate their function and understand their mechanisms.Wellcom

Novel stem cell technologies are powerful tools to understand the impact of human factors on Plasmodium falciparum malaria

Peer reviewed: TrueAcknowledgements: The authors wish to thank Marcus Lee for his overall support and Aleš Kilpatrick for helping with imaging.Plasmodium falciparum parasites have a complex life cycle, but the most clinically relevant stage of the disease is the invasion of erythrocytes and the proliferation of the parasite in the blood. The influence of human genetic traits on malaria has been known for a long time, however understanding the role of the proteins involved is hampered by the anuclear nature of erythrocytes that makes them inaccessible to genetic tools. Here we overcome this limitation using stem cells to generate erythroid cells with an in-vitro differentiation protocol and assess parasite invasion with an adaptation of flow cytometry to detect parasite hemozoin. We combine this strategy with reprogramming of patient cells to Induced Pluripotent Stem Cells and genome editing to understand the role of key genes and human traits in malaria infection. We show that deletion of basigin ablates invasion while deletion of ATP2B4 has a minor effect and that erythroid cells from reprogrammed patient-derived HbBart α-thalassemia samples poorly support infection. The possibility to obtain patient-secific and genetically modifed erythoid cells offers an unparalleled opportunity to study the role of human genes and polymorphisms in malaria allowing preservation of the genomic background to demonstrate their function and understand their mechanisms.</jats:p