Differential Plasmodium falciparum surface antigen expression among children with Malarial Retinopathy

Retinopathy provides a window into the underlying pathology of life-threatening malarial coma (“cerebral malaria”), allowing differentiation between 1) coma caused by sequestration of Plasmodium falciparum-infected erythrocytes in the brain and 2) coma with other underlying causes. Parasite sequestration in the brain is mediated by PfEMP1; a diverse parasite antigen that is inserted into the surface of infected erythrocytes and adheres to various host receptors. PfEMP1 sub-groups called “DC8” and “DC13” have been proposed to cause brain pathology through interactions with endothelial protein C receptor. To test this we profiled PfEMP1 gene expression in parasites from children with clinically defined cerebral malaria, who either had or did not have accompanying retinopathy. We found no evidence for an elevation of DC8 or DC13 PfEMP1 expression in children with retinopathy. However, the proportional expression of a broad subgroup of PfEMP1 called “group A” was elevated in retinopathy patients suggesting that these variants may play a role in the pathology of cerebral malaria. Interventions targeting group A PfEMP1 may be effective at reducing brain pathology

From contigs towards chromosomes: automatic improvement of long read assemblies (ILRA)

Recent advances in long read technologies not only enable large consortia to aim to sequence all eukaryotes on Earth, but they also allow individual laboratories to sequence their species of interest with relatively low investment. Long read technologies embody the promise of overcoming scaffolding problems associated with repeats and low complexity sequences, but the number of contigs often far exceeds the number of chromosomes and they may contain many insertion and deletion errors around homopolymer tracts. To overcome these issues, we have implemented the ILRA pipeline to correct long read-based assemblies. Contigs are first reordered, renamed, merged, circularized, or filtered if erroneous or contaminated. Illumina short reads are used subsequently to correct homopolymer errors. We successfully tested our approach by improving the genome sequences of Homo sapiens, Trypanosoma brucei, and Leptosphaeria spp., and by generating four novel Plasmodium falciparum assemblies from field samples. We found that correcting homopolymer tracts reduced the number of genes incorrectly annotated as pseudogenes, but an iterative approach seems to be required to correct more sequencing errors. In summary, we describe and benchmark the performance of our new tool, which improved the quality of novel long read assemblies up to 1 Gbp. The pipeline is available at GitHub: https://github.com/ThomasDOtto/ILRA

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

No evidence of P. falciparum K13 artemisinin conferring mutations over a 24-year analysis in Coastal Kenya, but a near complete reversion to chloroquine wild type parasites.

Antimalarial drug resistance is a substantial impediment to malaria control. The spread of resistance has been described using genetic markers which are important epidemiological tools. We carried out a temporal analysis of changes in allele frequencies of 12 drug resistance markers over two decades of changing antimalarial drug policy in Kenya. We did not detect any of the validated kelch 13 (k13) artemisinin resistance markers, nonetheless, a single k13 allele, K189T, was maintained at a stable high frequency (>10%) over time. There was a distinct shift from chloroquine resistant transporter (crt)-76, multi-drug resistant gene 1 (mdr1)-86 and mdr1-1246 chloroquine (CQ) resistance alleles to a 99% prevalence of CQ sensitive alleles in the population, following the withdrawal of CQ from routine use. In contrast, the dihydropteroate synthetase (dhps) double mutant (437G and 540E) associated with sulfadoxine-pyrimethamine (SP) resistance was maintained at a high frequency (>75%), after a change from SP to artemisinin combination therapies (ACTs). The novel cysteine desulfurase (nfs) K65 allele, implicated in resistance to lumefantrine in a West African study, showed a gradual significant decline in allele frequency pre- and post-ACT introduction (from 38% to 20%), suggesting evidence of directional selection in Kenya, potentially not due to lumefantrine. The high frequency of CQ-sensitive parasites circulating in the population suggests that the re-introduction of CQ in combination therapy for the treatment of malaria can be considered in the future. However, the risk of a re-emergence of CQ resistant parasites circulating below detectable levels or being reintroduced from other regions remains

Plasmodium falciparum malaria parasite var gene expression is modified by host antibodies: longitudinal evidence from controlled infections of Kenyan adults with varying natural exposure.

BACKGROUND: The PfEMP1 family of Plasmodium falciparum antigens play a key role in pathogenesis of severe malaria through their insertion into the surface of parasite infected erythrocytes, and adhesion to host cells. Previous studies have suggested that parasites expressing PfEMP1 subclasses group A and DC8, associated with severe malaria, may have a growth advantage in immunologically naïve individuals. However, this idea has not been tested in longitudinal studies. METHODS: Here we assessed expression of the var genes encoding PfEMP1, in parasites sampled from volunteers with varying prior exposure to malaria, following experimental infection by sporozoites (PfSPZ). Using qPCR, we tested for associations between the expression of various var subgroups in surviving parasite populations from each volunteer and 1) the levels of participants' antibodies to infected erythrocytes before challenge infection and 2) the apparent in vivo parasite multiplication rate. RESULTS: We show that 1) expression of var genes encoding for group A and DC8-like PfEMP1 were associated with low levels of antibodies to infected erythrocytes (αIE) before challenge, and 2) expression of a DC8-like CIDRα1.1 domain was associated with higher apparent parasite multiplication rate in a manner that was independent of levels of prior antibodies to infected erythrocytes. CONCLUSIONS: This study provides insight into the role of antibodies to infected erythrocytes surface antigens in the development of naturally acquired immunity and may help explain why specific PfEMP1 variants may be associated with severe malaria. TRIAL REGISTRATION: Pan African Clinical Trial Registry: PACTR201211000433272 . Date of registration: 10th October 2012

Exploring Plasmodium falciparum Var Gene Expression to Assess Host Selection Pressure on Parasites During Infancy

In sub-Saharan Africa, children below 5 years bear the greatest burden of severe malaria because they lack naturally acquired immunity that develops following repeated exposure to infections by Plasmodium falciparum. Antibodies to the surface of P. falciparum infected erythrocytes (IE) play an important role in this immunity. In children under the age of 6 months, relative protection from severe malaria is observed and this is thought to be partly due to trans-placental acquired protective maternal antibodies. However, the protective effect of maternal antibodies has not been fully established, especially the role of antibodies to variant surface antigens (VSA) expressed on IE. Here, we assessed the immune pressure on parasites infecting infants using markers associated with the acquisition of naturally acquired immunity to surface antigens. We hypothesized that, if maternal antibodies to VSA imposed a selection pressure on parasites, then the expression of a relatively conserved subset of var genes called group A var genes in infants should change with waning maternal antibodies. To test this, we compared their expression in parasites from children between 0 and 12 months and above 12 months of age. The transcript quantity and the proportional expression of group A var subgroup, including those containing domain cassette 13, were positively associated with age during the first year of life, which contrasts with above 12 months. This was accompanied by a decline in infected erythrocyte surface antibodies and an increase in parasitemia during this period. The observed increase in group A var gene expression with age in the first year of life, when the maternal antibodies are waning and before acquisition of naturally acquired antibodies with repeated exposure, is consistent with the idea that maternally acquired antibodies impose a selection pressure on parasites that infect infants and may play a role in protecting these infants against severe malaria

From contigs towards chromosomes: automatic improvement of long read assemblies (ILRA)

Recent advances in long read technologies not only enable large consortia to aim to sequence all eukaryotes on Earth, but they also allow individual laboratories to sequence their species of interest with relatively low investment. Long read technologies embody the promise of overcoming scaffolding problems associated with repeats and low complexity sequences, but the number of contigs often far exceeds the number of chromosomes and they may contain many insertion and deletion errors around homopolymer tracts. To overcome these issues, we have implemented the ILRA pipeline to correct long read-based assemblies. Contigs are first reordered, renamed, merged, circularized, or filtered if erroneous or contaminated. Illumina short reads are used subsequently to correct homopolymer errors. We successfully tested our approach by improving the genome sequences of Homo sapiens, Trypanosoma brucei, and Leptosphaeria spp., and by generating four novel Plasmodium falciparum assemblies from field samples. We found that correcting homopolymer tracts reduced the number of genes incorrectly annotated as pseudogenes, but an iterative approach seems to be required to correct more sequencing errors. In summary, we describe and benchmark the performance of our new tool, which improved the quality of novel long read assemblies up to 1 Gbp. The pipeline is available at GitHub: https://github.com/ThomasDOtto/ILRA

Plasmodium falciparum adapts its investment into replication versus transmission according to the host environment

The malaria parasite life cycle includes asexual replication in human blood, with a proportion of parasites differentiating to gametocytes required for transmission to mosquitoes. Commitment to differentiate into gametocytes, which is marked by activation of the parasite transcription factor ap2-g, is known to be influenced by host factors but a comprehensive model remains uncertain. Here we analyze data from 828 children in Kilifi, Kenya with severe, uncomplicated, and asymptomatic malaria infection over 18 years of falling malaria transmission. We examine markers of host immunity and metabolism, and markers of parasite growth and transmission investment. We find that inflammatory responses associated with reduced plasma lysophosphatidylcholine levels are associated with markers of increased investment in parasite sexual reproduction (i.e., transmission investment) and reduced growth (i.e., asexual replication). This association becomes stronger with falling transmission and suggests that parasites can rapidly respond to the within-host environment, which in turn is subject to changing transmission