Pathology Of Malaria Research Articles

Identification of novel PfEMP1 variants containing domain cassettes 11, 15 and 8 that mediate the Plasmodium falciparum virulence-associated rosetting phenotype.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a diverse family of variant surface antigens, encoded by var genes, that mediates binding of infected erythrocytes to human cells and plays a key role in parasite immune evasion and malaria pathology. The increased availability of parasite genome sequence data has revolutionised the study of PfEMP1 diversity across multiple P. falciparum isolates. However, making functional sense of genomic data relies on the ability to infer binding phenotype from var gene sequence. For P. falciparum rosetting, the binding of infected erythrocytes to uninfected erythrocytes, the analysis of var gene/PfEMP1 sequences encoding the phenotype is limited, with only eight rosette-mediating PfEMP1 variants described to date. These known rosetting PfEMP1 variants fall into two types, characterised by N-terminal domains known as "domain cassette" 11 (DC11) and DC16. Here we test the hypothesis that DC11 and DC16 are the only PfEMP1 types in the P. falciparum genome that mediate rosetting, by examining a set of thirteen recent culture-adapted Kenyan parasite lines. We first analysed the var gene/PfEMP1 repertoires of the Kenyan lines and identified an average of three DC11 or DC16 PfEMP1 variants per genotype. In vitro rosette selection of the parasite lines yielded four with a high rosette frequency, and analysis of their var gene transcription, infected erythrocyte PfEMP1 surface expression, rosette disruption and erythrocyte binding function identified four novel rosette-mediating PfEMP1 variants. Two of these were of the predicted DC11 type (one showing the dual rosetting/IgM-Fc-binding phenotype), whereas two contained DC15 (DBLα1.2-CIDRα1.5b) a PfEMP1 type not previously associated with rosetting. We also showed that a Thai parasite line expressing a DC8-like PfEMP1 binds to erythrocytes to form rosettes. Hence, these data expand current knowledge of rosetting mechanisms and emphasize that the PfEMP1 types mediating rosetting are more diverse than previously recognised.

Endothelial molecule levels (VCAM-1, ICAM-1) in uncomplicated malaria cases in Lagos, Nigeria

Background. Elevated endothelial activation markers are linked to malaria syndromes due to the sequestration of Plasmodium falciparum-infected red blood cells in deep tissue vessels. This study evaluated vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) levels in uncomplicated malaria and their association with host factors. Methods. A cross-sectional study was conducted in four health facilities in Ikorodu, Lagos State, Nigeria. Blood samples from children and adults were screened for malaria via microscopy, and plasma ICAM-1 and VCAM-1 levels were measured using ELISA. Results. ICAM-1 (1.03 × 106 ± 20,689.2 pg/ml) and VCAM-1 (1.11 × 106 pg/ml; range: 3,725–6,273,725 pg/ml) levels were significantly higher in malaria-positive cases compared to controls (p < 0.01). The geometric mean parasite density was 11,183 parasites/μl, but ICAM-1 and VCAM-1 levels did not correlate with parasite density (p > 0.1). Packed cell volume (PCV) was significantly lower in malaria-positive cases (p = 0.042), with ICAM-1 and VCAM-1 negatively correlating with PCV (p < 0.05). Both markers were also inversely associated with age (p < 0.05). Conclusion. VCAM-1 and ICAM-1 levels are elevated in uncomplicated malaria, independent of parasite density but negatively associated with PCV and age, highlighting the role of endothelial activation in malaria pathology.

Broadly inhibitory antibodies to severe malaria virulence proteins.

Malaria pathology is driven by the accumulation of Plasmodium falciparum-infected erythrocytes in microvessels1. This process is mediated by the polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins of the parasite. A subset of PfEMP1 variants that bind to human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis2. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here we describe two broadly reactive and inhibitory human monoclonal antibodies to CIDRα1. The antibodies isolated from two different individuals exhibited similar and consistent EPCR-binding inhibition of diverse CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins, as well as parasite sequestration in bioengineered 3D human brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with three different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies are likely to represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

The J Domain Proteins of Plasmodium knowlesi, a Zoonotic Malaria Parasite of Humans.

Plasmodium knowlesi is a zoonotic form of human malaria, the pathology of which is poorly understood. While the J domain protein (JDP) family has been extensively studied in Plasmodium falciparum, and shown to contribute to malaria pathology, there is currently very limited information on the P. knowlesi JDPs (PkJDPs). This review provides a critical analysis of the literature and publicly available data on PkJDPs. Interestingly, the P. knowlesi genome encodes at least 31 PkJDPs, with well over half belonging to the most diverse types which contain only the signature J domain (type IIIs, 19) or a corrupted version of the J domain (type IVs, 2) as evidence of their membership. The more typical PkJDPs containing other domains typical of JDPs in addition to the J domain are much fewer in number (type IIs, 8; type Is, 2). This study indentifies PkJDPs that are potentially involved in: folding of newly synthesized or misfolded proteins within the P. knowlesi cytosol (a canonical type I and certain typical type IIs); protein translocation (a type III) and folding (a type II) in the ER; and protein import into mitochondria (a type III). Interestingly, a type II PkJDP is potentially exported to the host cell cytosol where it may recruit human HSP70 for the trafficking and folding of other exported P. knowlesi proteins. Experimental studies are required on this fascinating family of proteins, not only to validate their role in the pathology of knowlesi malaria, but also because they represent potential anti-malarial drug targets.

Plasmodium falciparum protein phosphatase PP7 is required for early ring-stage development.

Plasmodium falciparum causes malaria and is responsible for more than 600,000 deaths each year. Although effective drugs are available to treat disease, the spread of drug-resistant parasites endangers their future efficacy. It is hoped that a better understanding of the biology of malaria parasites will help us to discover new drugs to tackle the resistance problem. Our work is focused on the cell signaling mechanisms that control the development of the parasite throughout its complex life cycle. All signal transduction pathways are ultimately regulated by reversible protein phosphorylation by protein kinase and protein phosphatase enzymes. In this study, we investigate the function of calcium-dependent protein phosphatase PP7 and show that it is essential for the development of ring-stage parasites following the invasion of human erythrocytes. Our results contribute to the understanding of the erythrocytic stages of the parasite life cycle that cause malaria pathology.

Upregulation of interferon gamma gene expression among asymptomatic malaria adults in Bayelsa State, Nigeria

Despite concerted efforts, malaria remains a global public health problem. Treatment for asymptomatic malaria will reduce the health care burden of malaria. However evidence-based data for the presence of systemic pathology in asymptomatic malaria, which might inform treatment remains limited. The present study investigated the current prevalence of asymptomatic malaria in Bayelsa State, Nigeria and differences in interferon gamma (IFNγ) mRNA expression between asymptomatic malaria individuals (AMIs) and uninfected individuals (UIs), in relation to ABO blood groups and hemoglobin genotype (Hb) variants. A cross-sectional design was employed to randomly select 146 non-febrile participants from Sagbama LGA, Bayelsa State. Plasmodium falciparum infection was determined using high-resolution melting analysis and participants were grouped into UIs and AMIs. Hb, blood groups, and IFNγ mRNA expression were determined using electrophoresis, agglutination, and real-time PCR method respectively. Logistic regression and t-test were used to determine significant differences between groups at p < 0.05. The current prevalence of asymptomatic malaria in Bayelsa State was found to be 89.73 %. Age and sex were associated with asymptomatic malaria (p < 0.05). Plasmodium falciparum DNA melt curves for AMIs aligned at 83.89 ± 0.34 °C, while the amplification cycle thresholds were below 30 cycles. Compared with UIs, the gene expression of IFNγ mRNA was significantly (p < 0.001) higher among AMIs. Also, among AMIs those with A, B, and AB blood groups expressed significantly higher levels of IFNγ mRNA compared with blood groups O AMIs. Furthermore, AMIs with HbAA expressed significantly higher levels of IFNγ mRNA compared with HbAS AMIs. The novelty of the present study is the demonstration of the existence of systemic pathology in asymptomatic malaria and the demonstration that the intensity of systemic pathology is dependent on blood types and haemoglobin genotype variants. Findings of the present study have implications for policy creation towards asymptomatic malaria treatment.

Comprehensive Analysis of Complex Copy Number Variations in the Plasmodium falciparum Genome via Long-Read Sequencing: Implications for Antimalarial Resistance and Broad Genomic Insights

Abstract Introduction/Objective The genome of Plasmodium falciparum, characterized by a high AT content and a propensity for copy number variations (CNVs), presents a model for understanding how genomes adapt to stress. Our research focused on the prevalence and distribution of CNVs highlights long-read sequencing as a tool for detecting complex structural variants in other models. Methods/Case Report P. falciparum parasites were cultured and treated with DSM1 and aphidicolin that induce replication stress. Long-read sequencing was conducted using the Oxford Nanopore Technology Minion, with an emphasis on an optimizing DNA extraction method for preserving intact chromosomes. Visualization of CNVs, including tandem and inverted duplications, was achieved through a custom R Shiny application that allowed both quantitative and qualitative assessments. Results (if a Case Study enter NA) Long read sequencing identified an enrichment of multiple CNV types in treated samples, with a significant enrichment of inverted duplications as well as other types of structural variants. Aphidicolin and DSM1-treated samples demonstrated significant accumulation of these variations compared to controls (2.0 and 5.6-fold, respectively with a p value of &amp;lt; 0.0001 in both treatment groups). These occurred mainly in core genomic regions that do not contain variable gene copies. An increase in the number of inverted duplications may be related to stress response to the treatment and may be a potential adaptive mechanism of the parasite. Our manual single-read visualization technique made it possible to visualize the structure of various CNV types, which provides unique insight on CNVs as they arise in single genomes. Conclusion We hypothesize that inverted duplications represent stalled replication forks, and we are currently assessing their frequency in replicating and non-replicating cells. This study highlights the role of long-read sequencing in detecting complex genomic structures in P. falciparum, which may influence the parasite’s resistance to drug treatment. These findings not only advance our understanding of malaria pathology but also aid our comprehension of how cancer cells might react to drug-induced stress and the mechanisms behind CNV formation in neoplastic cells.

APEX2-based proximity proteomic analysis identifies candidate interactors for Plasmodium falciparum knob-associated histidine-rich protein in infected erythrocytes

The interaction of Plasmodium falciparum—infected red blood cells (iRBCs) with the vascular endothelium plays a crucial role in malaria pathology and disease. KAHRP is an exported P. falciparum protein involved in iRBC remodelling, which is essential for the formation of protrusions or “knobs” on the iRBC surface. These knobs and the proteins that are concentrated within them allow the parasites to escape the immune response and host spleen clearance by mediating cytoadherence of the iRBC to the endothelial wall, but this also slows down blood circulation, leading in some cases to severe cerebral and placental complications. In this work, we have applied genetic and biochemical tools to identify proteins that interact with P. falciparum KAHRP using enhanced ascorbate peroxidase 2 (APEX2) proximity-dependent biotinylation and label-free shotgun proteomics. A total of 30 potential KAHRP-interacting candidates were identified, based on the assigned fragmented biotinylated ions. Several identified proteins have been previously reported to be part of the Maurer’s clefts and knobs, where KAHRP resides. This study may contribute to a broader understanding of P. falciparum protein trafficking and knob architecture and shows for the first time the feasibility of using APEX2-proximity labelling in iRBCs.

Different PfEMP1-expressing Plasmodium falciparum variants induce divergent endothelial transcriptional responses during co-culture.

The human malaria parasite Plasmodium falciparum is responsible for the majority of mortality and morbidity caused by malaria infection and differs from other human malaria species in the degree of accumulation of parasite-infected red blood cells in the microvasculature, known as cytoadherence or sequestration. In P. falciparum, cytoadherence is mediated by a protein called PfEMP1 which, due to its exposure to the host immune system, undergoes antigenic variation resulting in the expression of different PfEMP1 variants on the infected erythrocyte membrane. These PfEMP1s contain various combinations of adhesive domains, which allow for the differential engagement of a repertoire of endothelial receptors on the host microvasculature, with specific receptor usage associated with severe disease. We used a co-culture model of cytoadherence incubating human brain microvascular endothelial cells with erythrocytes infected with two parasite lines expressing different PfEMP1s that demonstrate different binding profiles to vascular endothelium. We determined the transcriptional profile of human brain microvascular endothelial cells (HBMEC) following different incubation periods with infected erythrocytes, identifying different transcriptional profiles of pathways previously found to be involved in the pathology of severe malaria, such as inflammation, apoptosis and barrier integrity, induced by the two PfEMP1 variants.

Association between Plasmodium Infection and Nitric Oxide Levels: A Systematic Review and Meta-Analysis.

Nitric oxide (NO) has been implicated in the pathology of malaria. This systematic review and meta-analysis describe the association between NO levels and malaria. Embase, Ovid, PubMed, Scopus, and Google Scholar were searched to identify studies evaluating NO levels in malaria patients and uninfected controls. Meta-regression and subgroup analyses were conducted to discern differences in NO levels between the groups. Of the 4517 records identified, 21 studies were included in the systematic review and meta-analysis. The findings illustrated significant disparities in NO levels based on geographic location and study time frames. Despite the fluctuations, such as higher NO levels in adults compared to children, no significant differences in mean NO levels between patients and uninfected controls (p = 0.25, Hedge's g: 0.35, 95% confidence interval (CI): -0.25-0.96, I2: 97.39%) or between severe and non-severe malaria cases (p = 0.09, Hedge's g: 0.71, 95% CI: -0.11-1.54, I2: 96.07%) were detected. The systematic review and meta-analysis highlighted inconsistencies in NO levels in malaria patients. Given the high heterogeneity of the results, further studies using standardized metrics for NO measurements and focusing on biochemical pathways dictating NO responses in malaria are imperative to understand the association between NO and malaria.

Cellular iron governs the host response to malaria.

Malaria and iron deficiency are major global health problems with extensive epidemiological overlap. Iron deficiency-induced anaemia can protect the host from malaria by limiting parasite growth. On the other hand, iron deficiency can significantly disrupt immune cell function. However, the impact of host cell iron scarcity beyond anaemia remains elusive in malaria. To address this, we employed a transgenic mouse model carrying a mutation in the transferrin receptor (TfrcY20H/Y20H), which limits the ability of cells to internalise iron from plasma. At homeostasis TfrcY20H/Y20H mice appear healthy and are not anaemic. However, TfrcY20H/Y20H mice infected with Plasmodium chabaudi chabaudi AS showed significantly higher peak parasitaemia and body weight loss. We found that TfrcY20H/Y20H mice displayed a similar trajectory of malaria-induced anaemia as wild-type mice, and elevated circulating iron did not increase peak parasitaemia. Instead, P. chabaudi infected TfrcY20H/Y20H mice had an impaired innate and adaptive immune response, marked by decreased cell proliferation and cytokine production. Moreover, we demonstrated that these immune cell impairments were cell-intrinsic, as ex vivo iron supplementation fully recovered CD4+ T cell and B cell function. Despite the inhibited immune response and increased parasitaemia, TfrcY20H/Y20H mice displayed mitigated liver damage, characterised by decreased parasite sequestration in the liver and an attenuated hepatic immune response. Together, these results show that host cell iron scarcity inhibits the immune response but prevents excessive hepatic tissue damage during malaria infection. These divergent effects shed light on the role of iron in the complex balance between protection and pathology in malaria.

Discrete class I molecules on brain endothelium differentially regulate neuropathology in experimental cerebral malaria.

Cerebral malaria is the deadliest complication that can arise from Plasmodium infection. CD8 T-cell engagement of brain vasculature is a putative mechanism of neuropathology in cerebral malaria. To define contributions of brain endothelial cell major histocompatibility complex (MHC) class I antigen-presentation to CD8 T cells in establishing cerebral malaria pathology, we developed novel H-2Kb LoxP and H-2Db LoxP mice crossed with Cdh5-Cre mice to achieve targeted deletion of discrete class I molecules, specifically from brain endothelium. This strategy allowed us to avoid off-target effects on iron homeostasis and class I-like molecules, which are known to perturb Plasmodium infection. This is the first endothelial-specific ablation of individual class-I molecules enabling us to interrogate these molecular interactions. In these studies, we interrogated human and mouse transcriptomics data to compare antigen presentation capacity during cerebral malaria. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM), we observed that H-2Kb and H-2Db class I molecules regulate distinct patterns of disease onset, CD8 T-cell infiltration, targeted cell death and regional blood-brain barrier disruption. Strikingly, ablation of either molecule from brain endothelial cells resulted in reduced CD8 T-cell activation, attenuated T-cell interaction with brain vasculature, lessened targeted cell death, preserved blood-brain barrier integrity and prevention of ECM and the death of the animal. We were able to show that these events were brain-specific through the use of parabiosis and created the novel technique of dual small animal MRI to simultaneously scan conjoined parabionts during infection. These data demonstrate that interactions of CD8 T cells with discrete MHC class I molecules on brain endothelium differentially regulate development of ECM neuropathology. Therefore, targeting MHC class I interactions therapeutically may hold potential for treatment of cases of severe malaria.

Non-O ABO blood group genotypes differ in their associations with Plasmodium falciparum rosetting and severe malaria.

Blood group O is associated with protection against severe malaria and reduced size and stability of P. falciparum-host red blood cell (RBC) rosettes compared to non-O blood groups. Whether the non-O blood groups encoded by the specific ABO genotypes AO, BO, AA, BB and AB differ in their associations with severe malaria and rosetting is unknown. The A and B antigens are host RBC receptors for rosetting, hence we hypothesized that the higher levels of A and/or B antigen on RBCs from AA, BB and AB genotypes compared to AO/BO genotypes could lead to larger rosettes, increased microvascular obstruction and higher risk of malaria pathology. We used a case-control study of Kenyan children and in vitro adhesion assays to test the hypothesis that "double dose" non-O genotypes (AA, BB, AB) are associated with increased risk of severe malaria and larger rosettes than "single dose" heterozygotes (AO, BO). In the case-control study, compared to OO, the double dose genotypes consistently had higher odds ratios (OR) for severe malaria than single dose genotypes, with AB (OR 1.93) and AO (OR 1.27) showing most marked difference (p = 0.02, Wald test). In vitro experiments with blood group A-preferring P. falciparum parasites showed that significantly larger rosettes were formed with AA and AB host RBCs compared to OO, whereas AO and BO genotypes rosettes were indistinguishable from OO. Overall, the data show that ABO genotype influences P. falciparum rosetting and support the hypothesis that double dose non-O genotypes confer a greater risk of severe malaria than AO/BO heterozygosity.

Xanthine oxidase levels and immune dysregulation are independently associated with anemia in Plasmodium falciparum malaria

Severe anemia is an important contributor to mortality in children with severe malaria. Anemia in malaria is a multi-factorial complication, since dyserythropoiesis, hemolysis and phagocytic clearance of uninfected red blood cells (RBCs) can contribute to this syndrome. High levels of oxidative stress and immune dysregulation have been proposed to contribute to severe malarial anemia, facilitating the clearance of uninfected RBCs. In a cohort of 552 Ugandan children with severe malaria, we measured the levels of xanthine oxidase (XO), an oxidative enzyme that is elevated in the plasma of malaria patients. The levels of XO in children with severe anemia were significantly higher compared to children with severe malaria not suffering from severe anemia. Levels of XO were inversely associated with RBC hemoglobin (ρ = − 0.25, p < 0.0001), indicating a relation between this enzyme and severe anemia. When compared with the levels of immune complexes and of autoimmune antibodies to phosphatidylserine, factors previously associated with severe anemia in malaria patients, we observed that XO is not associated with them, suggesting that XO is associated with severe anemia through an independent mechanism. XO was associated with prostration, acidosis, jaundice, respiratory distress, and kidney injury, which may reflect a broader relation of this enzyme with severe malaria pathology. Since inhibitors of XO are inexpensive and well-tolerated drugs already approved for use in humans, the validation of XO as a contributor to severe malarial anemia and other malaria complications may open new possibilities for much needed adjunctive therapy in malaria.

In silico identification of modulators of J domain protein-Hsp70 interactions in Plasmodium falciparum: a drug repurposing strategy against malaria.

Plasmodium falciparum is a unicellular, intracellular protozoan parasite, and the causative agent of malaria in humans, a deadly vector borne infectious disease. A key phase of malaria pathology, is the invasion of human erythrocytes, resulting in drastic remodeling by exported parasite proteins, including molecular chaperones and co-chaperones. The survival of the parasite within the human host is mediated by P. falciparum heat shock protein 70s (PfHsp70s) and J domain proteins (PfJDPs), functioning as chaperones-co-chaperones partnerships. Two complexes have been shown to be important for survival and pathology of the malaria parasite: PfHsp70-x-PFE0055c (exported); and PfHsp70-2-PfSec63 (endoplasmic reticulum). Virtual screening was conducted on the drug repurposing library, the Pandemic Response Box, to identify small-molecules that could specifically disrupt these chaperone complexes. Five top ranked compounds possessing preferential binding affinity for the malarial chaperone system compared to the human system, were identified; three top PfHsp70-PfJDP binders, MBX 1641, zoliflodacin and itraconazole; and two top J domain binders, ezetimibe and a benzo-diazepinone. These compounds were validated by repeat molecular dockings and molecular dynamics simulation, resulting in all the compounds, except for MBX 1461, being confirmed to bind preferentially to the malarial chaperone system. A detailed contact analysis of the PfHsp70-PfJDP binders identified two different types of modulators, those that potentially inhibit complex formation (MBX 1461), and those that potentially stabilize the complex (zoliflodacin and itraconazole). These data suggested that zoliflodacin and itraconazole are potential novel modulators specific to the malarial system. A detailed contact analysis of the J domain binders (ezetimibe and the benzo-diazepinone), revealed that they bound with not only greater affinity but also a better pose to the malarial J domain compared to that of the human system. These data suggested that ezetimibe and the benzo-diazepinone are potential specific inhibitors of the malarial chaperone system. Both itraconazole and ezetimibe are FDA-approved drugs, possess anti-malarial activity and have recently been repurposed for the treatment of cancer. This is the first time that such drug-like compounds have been identified as potential modulators of PfHsp70-PfJDP complexes, and they represent novel candidates for validation and development into anti-malarial drugs.

Novel secretory organelles of parasite origin - at the center of host-parasite interaction.

Reorganization of cell organelle-deprived host red blood cells by the apicomplexan malaria parasite Plasmodium falciparum enables their cytoadherence to endothelial cells that line the microvasculature. This increases the time red blood cells infected with mature developmental stages remain within selected organs such as the brain to avoid the spleen passage, which can lead to severe complications and cumulate in patient death. The Maurer's clefts are a novel secretory organelle of parasite origin established by the parasite in the cytoplasm of the host red blood cell in order to facilitate the establishment of cytoadherence by conducting the trafficking of immunovariant adhesins to the host cell surface. Another important function of the organelle is the sorting of other proteins the parasite traffics into its host cell. Although the organelle is of high importance for the pathology of malaria, additional putative functions, structure, and genesis remain shrouded in mystery more than a century after its discovery. In this review, we highlight our current knowledge about the Maurer's clefts and other novel secretory organelles established within the host cell cytoplasm by human-pathogenic malaria parasites and other parasites that reside within human red blood cells.

High throughput human genotyping for variants associated with malarial disease outcomes using custom targeted amplicon sequencing

Malaria has exhibited the strongest known selective pressure on the human genome in recent history and is the evolutionary driving force behind genetic conditions, such as sickle-cell disease, glucose-6-phosphatase deficiency, and some other erythrocyte defects. Genomic studies (e.g., The 1000 Genomes project) have provided an invaluable baseline for human genetics, but with an estimated two thousand ethno-linguistic groups thought to exist across the African continent, our understanding of the genetic differences between indigenous populations and their implications on disease is still limited. Low-cost sequencing-based approaches make it possible to target specific molecular markers and genes of interest, leading to potential insights into genetic diversity. Here we demonstrate the versatility of custom dual-indexing technology and Illumina next generation sequencing to generate a genetic profile of human polymorphisms associated with malaria pathology. For 100 individuals diagnosed with severe malaria in Northeast Tanzania, variants were successfully characterised on the haemoglobin subunit beta (HBB), glucose-6-phosphate dehydrogenase (G6PD), atypical chemokine receptor 1 (ACKR1) genes, and the intergenic Dantu genetic blood variant, then validated using pre-existing genotyping data. High sequencing coverage was observed across all amplicon targets in HBB, G6PD, ACKR1, and the Dantu blood group, with variants identified at frequencies previously observed within this region of Tanzania. Sequencing data exhibited high concordance rates to pre-existing genotyping data (> 99.5%). Our work demonstrates the potential utility of amplicon sequencing for applications in human genetics, including to personalise medicine and understand the genetic diversity of loci linked to important host phenotypes, such as malaria susceptibility.

Roles of the RON3 C-terminal fragment in erythrocyte invasion and blood-stage parasite proliferation in Plasmodium falciparum.

Plasmodium species cause malaria, and in the instance of Plasmodium falciparum is responsible for a societal burden of over 600,000 deaths annually. The symptoms and pathology of malaria are due to intraerythocytic parasites. Erythrocyte invasion is mediated by the parasite merozoite stage, and is accompanied by the formation of a parasitophorous vacuolar membrane (PVM), within which the parasite develops. The merozoite apical rhoptry organelle contains various proteins that contribute to erythrocyte attachment and invasion. RON3, a rhoptry bulb membrane protein, undergoes protein processing and is discharged into the PVM during invasion. RON3-deficient parasites fail to develop beyond the intraerythrocytic ring stage, and protein export into erythrocytes by the Plasmodium translocon of exported proteins (PTEX) apparatus is abrogated, as well as glucose uptake into parasites. It is known that truncated N- and C-terminal RON3 fragments are present in rhoptries, but it is unclear which RON3 fragments contribute to protein export by PTEX and glucose uptake through the PVM. To investigate and distinguish the roles of the RON3 C-terminal fragment at distinct developmental stages, we used a C-terminus tag for conditional and post-translational control. We demonstrated that RON3 is essential for blood-stage parasite survival, and knockdown of RON3 C-terminal fragment expression from the early schizont stage induces a defect in erythrocyte invasion and the subsequent development of ring stage parasites. Protein processing of full-length RON3 was partially inhibited in the schizont stage, and the RON3 C-terminal fragment was abolished in subsequent ring-stage parasites compared to the RON3 N-terminal fragment. Protein export and glucose uptake were abrogated specifically in the late ring stage. Plasmodial surface anion channel (PSAC) activity was partially retained, facilitating small molecule traffic across the erythrocyte membrane. The knockdown of the RON3 C-terminal fragment after erythrocyte invasion did not alter parasite growth. These data suggest that the RON3 C-terminal fragment participates in erythrocyte invasion and serves an essential role in the progression of ring-stage parasite growth by the establishment of the nutrient-permeable channel in the PVM, accompanying the transport of ring-stage parasite protein from the plasma membrane to the PVM.

Plasmodium falciparum GAP40 Plays an Essential Role in Merozoite Invasion and Gametocytogenesis.

Cyclic invasion of red blood cells (RBCs) by Plasmodium merozoites is associated with the symptoms and pathology of malaria. Merozoite invasion is powered actively and rapidly by a parasite actomyosin motor called the glideosome. The ability of the glideosome to generate force to support merozoite entry into the host RBCs is thought to rely on its stable anchoring within the inner membrane complex (IMC) through membrane-resident proteins, such as GAP50 and GAP40. Using a conditional knockdown (KD) approach, we determined that PfGAP40 was required for asexual blood-stage replication. PfGAP40 is not needed for merozoite egress from host RBCs or for the attachment of merozoites to new RBCs. PfGAP40 coprecipitates with PfGAP45 and PfGAP50. During merozoite invasion, PfGAP40 is associated strongly with stabilizing the expression levels of PfGAP45 and PfGAP50 in the schizont stage. Although PfGAP40 KD did not influence IMC integrity, it impaired the maturation of gametocytes. In addition, PfGAP40 is phosphorylated, and mutations that block phosphorylation of PfGAP40 at the C-terminal serine residues S370, S372, S376, S405, S409, S420, and S445 reduced merozoite invasion efficiency. Overall, our findings implicate PfGAP40 as an important regulator for the gliding activity of merozoites and suggest that phosphorylation is required for PfGAP40 function. IMPORTANCE Red blood cell invasion is central to the pathogenesis of the malaria parasite, and the parasite proteins involved in this process are potential therapeutic targets. Gliding motility powers merozoite invasion and is driven by a unique molecular motor termed the glideosome. The glideosome is stably anchored to the parasite inner membrane complex (IMC) through membrane-resident proteins. In the present study, we demonstrate the importance of an IMC-resident glideosome component, PfGAP40, that plays a critical role in stabilizing the expression levels of glideosome components in the schizont stage. We determined that phosphorylation of PfGAP40 at C-terminal residues is required for efficient merozoite invasion.

Plasmodium falciparum-infected erythrocyte co-culture with the monocyte cell line THP-1 does not trigger production of soluble factors reducing brain microvascular barrier function.

Monocytes contribute to the pro-inflammatory immune response during the blood stage of a Plasmodium falciparum infection, but their precise role in malaria pathology is not clear. Besides phagocytosis, monocytes are activated by products from P. falciparum infected erythrocytes (IE) and one of the activation pathways is potentially the NLR family pyrin domain containing 3 (NLRP3) inflammasome, a multi-protein complex that leads to the production of interleukin (IL)-1β. In cerebral malaria cases, monocytes accumulate at IE sequestration sites in the brain microvascular and the locally produced IL-1β, or other secreted molecules, could contribute to leakage of the blood-brain barrier. To study the activation of monocytes by IE within the brain microvasculature in an in vitro model, we co-cultured IT4var14 IE and the monocyte cell line THP-1 for 24 hours and determined whether generated soluble molecules affect barrier function of human brain microvascular endothelial cells, measured by real time trans-endothelial electrical resistance. The medium produced after co-culture did not affect endothelial barrier function and similarly no effect was measured after inducing oxidative stress by adding xanthine oxidase to the co-culture. While IL-1β does decrease barrier function, barely any IL-1β was produced in the co- cultures, indicative of a lack of or incomplete THP-1 activation by IE in this co-culture model.