Cerebral malaria: insight into pathology from optical coherence tomography.

We aimed to investigate structural retinal changes in malarial retinopathy (MR) using hand-held optical coherence tomography (HH-OCT) to assess its diagnostic potential. Children with MR (n = 43) underwent ophthalmoscopy, fluorescein angiography and HH-OCT during admission, 1-month (n = 31) and 1-year (n = 8) post-discharge. Controls were comatose patients without malaria (n = 6) and age/sex-matched healthy children (n = 43). OCT changes and retinal layer thicknesses were compared. On HH-OCT, hyper-reflective areas (HRAs) were seen in the inner retina of 81% of MR patients, corresponding to ischaemic retinal whitening on fundus photography. Cotton wool spots were present in 37% and abnormal hyper-reflective dots, co-localized to capillary plexus, in 93%. Hyper-reflective vessel walls were present in 84%, and intra-retinal cysts in 9%. Vascular changes and cysts resolved within 48 h. HRAs developed into retinal thinning at 1 month (p = 0.027) which was more pronounced after 1 year (p = 0.009). Ischaemic retinal whitening is located within inner retinal layers, distinguishing it from cotton wool spots. Vascular hyper-reflectivity may represent the sequestration of parasitized erythrocytes in vessels, a key CM feature. The mechanisms of post-ischemic retinal atrophy and cerebral atrophy with cognitive impairment may be similar in CM survivors. HH-OCT has the potential for monitoring patients, treatment response and predicting neurological deficits.

Inflammatory cytokine responses in children with asymptomatic malaria infection living in rural, semi-urban and urban areas in south-eastern Gabon.

Cytokines are soluble mediators of the immune response, and their evolution influences the disease outcome. Gaining knowledge on cytokines has become important, as they can constitute biomarkers allowing the diagnosis of malaria and preventing severe forms of the disease. Here, we investigated 10 cytokines and their circulating levels in asymptomatic Gabonese children with Plasmodium falciparum infection living in urban, semi-urban and rural areas. Blood samples were collected from 273 schoolchildren (153 uninfected and 120 infected) aged 6 to 192 months. Hematological parameters were determined and P. falciparum diagnosis was performed using a rapid diagnosis test, microscopy and nested polymerase chain reaction (PCR). Plasma pro- [interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12p70, IL-17A and IL-22] and anti-inflammatory [IL-10, IL-4, IL-13 and transforming growth factor (TGF)-ß] cytokine levels were measured by enzyme-linked immunosorbent assay (ELISA) and compared between asymptomatic-infected and uninfected children. Results revealed that without distinction of area, IL-10 and IL-6 levels were higher in infected compared to uninfected children; however, the pro- and anti-inflammatory ratios (IL-6/IL-10 and TNF-α/IL-10) were similar. Furthermore, with area distinction significantly elevated levels of IL-10 in these asymptomatic children were always accompanied by either significantly low or high levels of a proinflammatory cytokine. Also, comparison between asymptomatic-infected children from the three areas showed significantly lower IL-17A, IL-22 and TGF-ß levels in urban area compared to semi-urban and rural areas. These results suggest that asymptomatic malaria infections induce significantly high inflammatory cytokine levels without modifying the balanced between pro- and anti-inflammatory cytokines and underline the higher exposure to infections of children in rural areas.

Positron emission tomography and magnetic resonance imaging in experimental human malaria to identify organ-specific changes in morphology and glucose metabolism: A prospective cohort study.

BACKGROUND: Plasmodium vivax has been proposed to infect and replicate in the human spleen and bone marrow. Compared to Plasmodium falciparum, which is known to undergo microvascular tissue sequestration, little is known about the behavior of P. vivax outside of the circulating compartment. This may be due in part to difficulties in studying parasite location and activity in life. METHODS AND FINDINGS: To identify organ-specific changes during the early stages of P. vivax infection, we performed 18-F fluorodeoxyglucose (FDG) positron emission tomography/magnetic resonance imaging (PET/MRI) at baseline and just prior to onset of clinical illness in P. vivax experimentally induced blood-stage malaria (IBSM) and compared findings to P. falciparum IBSM. Seven healthy, malaria-naive participants were enrolled from 3 IBSM trials: NCT02867059, ACTRN12616000174482, and ACTRN12619001085167. Imaging took place between 2016 and 2019 at the Herston Imaging Research Facility, Australia. Postinoculation imaging was performed after a median of 9 days in both species (n = 3 P. vivax; n = 4 P. falciparum). All participants were aged between 19 and 23 years, and 6/7 were male. Splenic volume (P. vivax: +28.8% [confidence interval (CI) +10.3% to +57.3%], P. falciparum: +22.9 [CI -15.3% to +61.1%]) and radiotracer uptake (P. vivax: +15.5% [CI -0.7% to +31.7%], P. falciparum: +5.5% [CI +1.4% to +9.6%]) increased following infection with each species, but more so in P. vivax infection (volume: p = 0.72, radiotracer uptake: p = 0.036). There was no change in FDG uptake in the bone marrow (P. vivax: +4.6% [CI -15.9% to +25.0%], P. falciparum: +3.2% [CI -3.2% to +9.6%]) or liver (P. vivax: +6.2% [CI -8.7% to +21.1%], P. falciparum: -1.4% [CI -4.6% to +1.8%]) following infection with either species. In participants with P. vivax, hemoglobin, hematocrit, and platelet count decreased from baseline at the time of postinoculation imaging. Decrements in hemoglobin and hematocrit were significantly greater in participants with P. vivax infection compared to P. falciparum. The main limitations of this study are the small sample size and the inability of this tracer to differentiate between host and parasite metabolic activity. CONCLUSIONS: PET/MRI indicated greater splenic tropism and metabolic activity in early P. vivax infection compared to P. falciparum, supporting the hypothesis of splenic accumulation of P. vivax very early in infection. The absence of uptake in the bone marrow and liver suggests that, at least in early infection, these tissues do not harbor a large parasite biomass or do not provoke a prominent metabolic response. PET/MRI is a safe and noninvasive method to evaluate infection-associated organ changes in morphology and glucose metabolism.

[Fe-S] biogenesis and unusual assembly of the ISC scaffold complex in the Plasmodium falciparum mitochondrion.

The malaria parasite harbors two [Fe-S] biogenesis pathways of prokaryotic origin-the SUF and ISC systems in the apicoplast and mitochondrion, respectively. While the SUF machinery has been delineated, there is little experimental evidence on the ISC pathway. We confirmed mitochondrial targeting of Plasmodium falciparum ISC proteins followed by analyses of cysteine desulfurase, scaffold, and [Fe-S]-carrier components. PfIscU functioned as the scaffold in complex with the PfIscS-PfIsd11 cysteine desulfurase and could directly assemble [4Fe-4S] without prior [2Fe-2S] formation seen in other homologs. Small angle X-ray scattering and spectral studies showed that PfIscU, a trimer, bound one [4Fe-4S]. In a deviation from reported complexes from other organisms, the P. falciparum desulfurase-scaffold complex assembled around a PfIscS tetramer instead of a dimer, resulting in a symmetric hetero-hexamer [2× (2PfIscS-2PfIsd11-2PfIscU)]. PfIscU directly transferred [4Fe-4S] to the apo-protein aconitase B thus abrogating the requirement of intermediary proteins for conversion of [2Fe-2S] to [4Fe-4S] before transfer to [4Fe-4S]-recipients. Among the putative cluster-carriers, PfIscA2 was more efficient than PfNifU-like protein; PfIscA1 primarily bound iron, suggesting its potential role as a Fe2+ carrier/donor. Our results identify the core P. falciparum ISC machinery and reveal unique features compared with those in bacteria or yeast and human mitochondria.

An integrated virtual screening and drug repurposing strategy for the discovery of new antimalarial drugs against Plasmodium falciparum phosphatidylinositol 3-kinase.

The emergence and spread of drug resistance in Plasmodium falciparum, the parasite causing the most severe form of human malaria, is a major threat to malaria control and elimination programs around the globe. With P. falciparum having evolved widespread resistance against a number of previously widely used drugs, currently, artemisinin (ART) and its derivatives are the cornerstones of first-line treatments of uncomplicated malaria. However, growing incidences of ART failure reflect the spread of ART-resistant P. falciparum strains. Despite current efforts to understand the primary cause of ART resistance due to mutations in the Kelch 13 gene (PfK13), the mechanism underlying ART resistance is still not completely unclear and no feasible strategies to counteract the causes and thereby restoring the efficiency of ART have been developed. We use a polypharmacology approach to identify potential drugs that can be used for the novel purpose (target). Of note, we have designed a multimodal stratagem to identify approved drugs with a potential antimalarial activity using computational drug reprofiling. Our investigations suggest that oxetacaine, simvastatin, repaglinide, aclidinium, propafenone, and lovastatin could be repurposed for malaria control and prevention.

Diversity and expression of Plasmodium falciparum var gene in severe and mild malaria cases from Central India.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein is encoded by a highly variable multicopy var gene family known to play a key role in malaria pathogenicity. Therefore, we investigated sequence variation, expression profile and immune response of the Duffy binding-like domain (DBLα) region of the var gene. METHODS: Blood samples were collected from patients with cerebral, severe and mild malaria in Chhattisgarh, India, a region with endemic malaria. Polymerase chain reaction amplicons were cloned and sequenced to determine sequence variation. The expression level was analyzed targeting the upstream region of var gene using the Delta-Delta-Ct method. Immunoglobulin G (IgG) level was determined against the 6 synthetic peptides of the DBLα region. RESULTS: The study identified that group 1 and group 5 sequences (cysteine/position of limited variability (cys/PoLV) classification) along with cys2/cys4 and MFK*/REY motifs and short amino acid length were significantly associated with malaria severity. The specific PoLV (MFKS, LREA, PTNL) were restricted to cerebral malaria. The expression level of var group A was higher than var groups B and C, demonstrating its prognostic characteristic. All peptides showed high-quality IgG response, while VAR P5 appeared to be a good marker for severity. CONCLUSIONS: The present study illustrates the presence of specific sequences of DBLα tags involved in severe malaria that could be targeted in future interventions for malaria control and elimination.

Depletion of cholesterol could be associated with modulation of progesterone but not other sex hormone levels during Plasmodium falciparum infection in humans: a cross-sectional study from Zaria, Nigeria.

In order for Plasmodium falciparum to grow and survive in its host, membrane biogenesis, fueled by host cholesterol, is essential for these processes. Consistent with this essential role, more insights into the cholesterol pathway would enhance the current understanding of the pathophysiology of malaria infection. To explore its broader potential, we conducted a cross-sectional study and assayed for the serum levels of cholesterol, vitamin D, progesterone, testosterone, estradiol and bile acid in both P. falciparum-infected patients and apparently healthy sex-matched participants. Our results revealed that the levels of cholesterol, vitamin D, progesterone, testosterone and estradiol in P. falciparum-infected patients were significantly (p < 0.05) lower compared to those in control groups whereas the level of bile acid in P. falciparum-infected patients was significantly (p < 0.05) higher compared to that in control groups. Additionally, cholesterol and the metabolic products with the exception of bile acid had a significant (p < 0.05) association with the parasite density in P. falciparum-infected patients with moderate and high P. falciparum infections. Furthermore, all the metabolic products of cholesterol had an insignificant (p > 0.05) association with the cholesterol in P. falciparum-infected patients with the exception of progesterone which showed a significant (p < 0.05) association with cholesterol in the malaria-infected female patients. Data from the present study demonstrated that progesterone depletion in P. falciparum-infected female patients could be a consequence of P. falciparum-induced decrease in cholesterol.

Inflammasome activation and IL-1 signaling during placental malaria induce poor pregnancy outcomes.

Placental malaria (PM) is associated with severe inflammation leading to abortion, preterm delivery, and intrauterine growth restriction. Innate immunity responses play critical roles, but the mechanisms underlying placental immunopathology are still unclear. Here, we investigated the role of inflammasome activation in PM by scrutinizing human placenta samples from an endemic area and ablating inflammasome components in a PM mouse model. The reduction in birth weight in babies from infected mothers is paralleled by increased placental expression of AIM2 and NLRP3 inflammasomes. Using genetic dissection, we reveal that inflammasome activation pathways are involved in the production and detrimental action of interleukin-1ß (IL-1ß) in the infected placenta. The IL-1R pharmacological antagonist Anakinra improved pregnancy outcomes by restoring fetal growth and reducing resorption in an experimental model. These findings unveil that IL-1ß-mediated signaling is a determinant of PM pathogenesis, suggesting that IL-1R antagonists can improve clinical outcomes of malaria infection in pregnancy.

The activation of BAFF/APRIL system in spleen and lymph nodes of Plasmodium falciparum infected patients.

Previous studies have reported activation of the B cell-activating factor (BAFF)/a proliferation-inducing ligand (APRIL) system in T independent immunity against malaria infection. Plasmodium falciparum (P. falciparum) infected animal model is not feasible. Therefore, little is known about the occurrence of BAFF/APRIL system and changes in falciparum lymphoid tissues. This study aimed to investigate the expression of BAFF/APRIL system components in lymphoid tissues from P. falciparum infected patients. Spleen and lymph node samples from 14 patients were collected at autopsy. Normal spleens and bacterially infected tonsils served as controls. The protein and/or mRNA expression of BAFF/APRIL and their cognate receptors, BAFF-R, TACI and BCMA, were determined by immunohistochemistry and RT-qPCR, respectively. The spleens of the patients exhibited significantly higher BAFF-R protein expression than normal spleens. Although without appropriate control, BCMA protein was markedly observed only in the lymph nodes. BAFF and BCMA mRNA levels were also significantly elevated in the spleen tissues of the patients compared with normal spleens. The overall BAFF-R protein levels in the lymphoid tissues of the patients correlated positively with parasitaemia. These findings are the first to confirm that BAFF/APRIL system activation in lymphoid tissues and is positively correlated with the parasitaemia levels in falciparum malaria.

A novel murine model for assessing fetal and birth outcomes following transgestational maternal malaria infection.

Plasmodium falciparum infection during pregnancy is a major cause of severe maternal illness and neonatal mortality. Mouse models are important for the study of gestational malaria pathogenesis. When infected with Plasmodium chabaudi chabaudi AS in early gestation, several inbred mouse strains abort at midgestation. We report here that outbred Swiss Webster mice infected with P. chabaudi chabaudi AS in early gestation carry their pregnancies to term despite high parasite burden and malarial hemozoin accumulation in the placenta at midgestation, with the latter associated with induction of heme oxygenase 1 expression. Infection yields reduced fetal weight and viability at term and a reduction in pup number at weaning, but does not influence postnatal growth prior to weaning. This novel model allows for the exploration of malaria infection throughout pregnancy, modeling chronic infections observed in pregnant women prior to the birth of underweight infants and enabling the production of progeny exposed to malaria in utero, which is critical for understanding the postnatal repercussions of gestational malaria. The use of outbred mice allows for the exploration of gestational malaria in a genetically diverse model system, better recapitulating the diversity of infection responses observed in human populations.

Primaquine homodimers as potential antiplasmodial and anticancer agents.

Primaquine homodimers, e.g. symmetric PQ-diamides of dicarboxylic acids containing 4 to 8 carbon atoms, were evaluated against Plasmodium berghei hepatic stages and P. falciparum blood stages, as well as against three cancer cell lines. Novel PQ-homodimers exerted much higher activity against hepatic stages, but less pronounced activity against blood stages in comparison to the parent drug. The submicromolar activity of succinic, fumaric and maleic derivatives against P. berghei was determined (IC50 values: 726.2, 198.1 and 358.4 nM, respectively). Our results indicated that the length and type of spacer between two PQ moieties highly modified the antiproliferative activities of PQ-homodimers. The general antiproliferative activity of the adipic and mesaconic derivatives against three cancer cell lines (MCF-7, HCT116, H 460) was observed (GI50 = 1.78-13.7 and 2.36-4.31 µM, respectively), but adipic derivative was less toxic to human embryonic kidney cells (HEK 293). High selectivity of fumaric and suberic derivatives against breast adenocarcinoma cell line MCF-7 was detected. These two compounds have shown no antiproliferative activity against other tumor cells and HEK 293.

Generation of an immortalized erythroid progenitor cell line from peripheral blood: A model system for the functional analysis of Plasmodium spp. invasion.

Malaria pathogenesis is caused by the replication of Plasmodium parasites within the red blood cells (RBCs) of the vertebrate host. This selective pressure has favored the evolution of protective polymorphisms in erythrocyte proteins, a subset of which serve as cognate receptors for parasite invasion ligands. Recently, the generation of RBCs from immortalized hematopoietic stem cells (HSCs) has offered a more tractable system for genetic manipulation and long-term in vitro culture, enabling elucidation of the functional determinants of host susceptibility in vitro. Here we report the generation of an immortalized erythroid progenitor cell line (EJ cells) from as few as 100 000 peripheral blood mononuclear cells. It offers a robust method for the creation of customized model systems from small volumes of peripheral blood. The EJ cell differentiation mirrored erythropoiesis of primary HSCs, yielding orthochromatic erythroblasts and enucleated RBCs after eight days (ejRBCs). The ejRBCs supported invasion by both P. vivax and P. falciparum. To demonstrate the genetic tractability of this system, we used CRISPR/Cas9 to disrupt the Duffy Antigen/Receptor for Chemokines (DARC) gene, which encodes the canonical receptor of P. vivax in humans. Invasion of P. vivax into this DARC-knockout cell line was strongly inhibited providing direct genetic evidence that P. vivax requires DARC for RBC invasion. Further, genetic complementation of DARC restored P. vivax invasion. Taken together, the peripheral blood immortalization method presented here offers the capacity to generate biologically representative model systems for studies of blood-stage malaria invasion from the peripheral blood of donors harboring unique genetic backgrounds, or rare polymorphisms.

Prime and target immunization protects against liver-stage malaria in mice.

Despite recent advances in treatment and vector control, malaria is still a leading cause of death, emphasizing the need for an effective vaccine. The malaria life cycle can be subdivided into three stages: the invasion and growth within liver hepatocytes (pre-erythrocytic stage), the blood stage (erythrocytic stage), and, finally, the sexual stage (occurring within the mosquito vector). Antigen (Ag)-specific CD8+ T cells are effectively induced by heterologous prime-boost viral vector immunization and known to correlate with liver-stage protection. However, liver-stage malaria vaccines have struggled to generate and maintain the high numbers of Plasmodium-specific circulating T cells necessary to confer sterile protection. We describe an alternative "prime and target" vaccination strategy aimed specifically at inducing high numbers of tissue-resident memory T cells present in the liver at the time of hepatic infection. This approach bypasses the need for very high numbers of circulating T cells and markedly increases the efficacy of subunit immunization against liver-stage malaria with clinically relevant Ags and clinically tested viral vectors in murine challenge models. Translation to clinical use has begun, with encouraging results from a pilot safety and feasibility trial of intravenous chimpanzee adenovirus vaccination in humans. This work highlights the value of a prime-target approach for immunization against malaria and suggests that this strategy may represent a more general approach for prophylaxis or immunotherapy of other liver infections and diseases.

Targeting the IL33-NLRP3 axis improves therapy for experimental cerebral malaria.

Cerebral malaria (CM) is a serious neurological complication caused by Plasmodium falciparum infection. Currently, the only treatment for CM is the provision of antimalarial drugs; however, such treatment by itself often fails to prevent death or development of neurological sequelae. To identify potential improved treatments for CM, we performed a nonbiased whole-brain transcriptomic time-course analysis of antimalarial drug chemotherapy of murine experimental CM (ECM). Bioinformatics analyses revealed IL33 as a critical regulator of neuroinflammation and cerebral pathology that is down-regulated in the brain during fatal ECM and in the acute period following treatment of ECM. Consistent with this, administration of IL33 alongside antimalarial drugs significantly improved the treatment success of established ECM. Mechanistically, IL33 treatment reduced inflammasome activation and IL1ß production in microglia and intracerebral monocytes in the acute recovery period following treatment of ECM. Moreover, treatment with the NLRP3-inflammasome inhibitor MCC950 alongside antimalarial drugs phenocopied the protective effect of IL33 therapy in improving the recovery from established ECM. We further showed that IL1ß release from macrophages was stimulated by hemozoin and antimalarial drugs and that this was inhibited by MCC950. Our results therefore demonstrate that manipulation of the IL33-NLRP3 axis may be an effective therapy to suppress neuroinflammation and improve the efficacy of antimalarial drug treatment of CM.

Plasmodium falciparum dipeptidyl aminopeptidase 3 activity is important for efficient erythrocyte invasion by the malaria parasite.

Parasite egress from infected erythrocytes and invasion of new red blood cells are essential processes for the exponential asexual replication of the malaria parasite. These two tightly coordinated events take place in less than a minute and are in part regulated and mediated by proteases. Dipeptidyl aminopeptidases (DPAPs) are papain-fold cysteine proteases that cleave dipeptides from the N-terminus of protein substrates. DPAP3 was previously suggested to play an essential role in parasite egress. However, little is known about its enzymatic activity, intracellular localization, or biological function. In this study, we recombinantly expressed DPAP3 and demonstrate that it has indeed dipeptidyl aminopeptidase activity, but contrary to previously studied DPAPs, removal of its internal prodomain is not required for activation. By combining super resolution microscopy, time-lapse fluorescence microscopy, and immunoelectron microscopy, we show that Plasmodium falciparum DPAP3 localizes to apical organelles that are closely associated with the neck of the rhoptries, and from which DPAP3 is secreted immediately before parasite egress. Using a conditional knockout approach coupled to complementation studies with wild type or mutant DPAP3, we show that DPAP3 activity is important for parasite proliferation and critical for efficient red blood cell invasion. We also demonstrate that DPAP3 does not play a role in parasite egress, and that the block in egress phenotype previously reported for DPAP3 inhibitors is due to off target or toxicity effects. Finally, using a flow cytometry assay to differentiate intracellular parasites from extracellular parasites attached to the erythrocyte surface, we show that DPAP3 is involved in the initial attachment of parasites to the red blood cell surface. Overall, this study establishes the presence of a DPAP3-dependent invasion pathway in malaria parasites.

Featured Article: Immunomodulatory effect of hemozoin on pneumocyte apoptosis via CARD9 pathway, a possibly retarding pulmonary resolution.

Plasmodium falciparum, the most virulent malaria parasite species, causes severe symptoms especially acute lung injury (ALI), of which characterized by alveolar epithelium and endothelium destruction and accelerated to blood-gas-barrier breakdown. Parasitized erythrocytes, endothelial cells, monocytes, and cytokines are all involved in this mechanism, but hemozoin (HZ), the parasitic waste from heme detoxification, also mainly contributes. In addition, it is not clear why type II pneumocyte proliferation, alveolar restorative stage, is rare in malaria-associated ALI. To address this, in vitro culture of A549 cells with Plasmodium HZ or with interleukin (IL)-1ß triggered by HZ and monocytes (HZ-IL-1ß) was conducted to determine their alveolar apoptotic effect using ethidium bromide/acridine orange staining, annexin-V-FITC/propidium iodide staining, and electron mircroscopic study. Caspase recruitment domain-containing protein 9 ( CARD9), the apoptotic regulator gene, and IL-1ß were quantified by reverse-transcriptase PCR. Junctional cellular defects were characterized by immunohistochemical staining of E-cadherin. The results revealed that cellular apoptosis and CARD9 expression levels were extremely high 24 h after induction by HZ-IL-1ß when compared to the HZ- and non-treated groups. E-cadherin was markedly down-regulated by HZ-IL-1ß and HZ treatments. CARD9 expression was positively correlated with IL-1ß expression and the number of apoptotic cells. Interestingly, the localization of HZ in the vesicular surfactant of apoptotic pneumocyte was also identified and submitted to be a cause of alveolar resolution abnormality. Thus, HZ triggers monocytes to produce IL-1ß and induces pneumocyte type II apoptosis through CARD9 pathway in association with down-regulated E-cadherin, which probably impairs alveolar resolution in malaria-associated ALI. Impact statement The present work shows the physical and immunomodulatory properties of hemozoin on the induction of pneumocyte apoptosis in relation to IL-1ß production through the CARD9 pathway. This occurrence may be a possible pathway for the retardation of lung resolution leading to blood-gas-barrier breakdown. Our findings lead to the understanding of the host-parasite relationship focusing on the dysfunction in ALI induced by HZ, a possible pathway of the recovering lung epithelial retardation in malaria-associated ARDS.

A protease cascade regulates release of the human malaria parasite Plasmodium falciparum from host red blood cells.

Malaria parasites replicate within a parasitophorous vacuole in red blood cells (RBCs). Progeny merozoites egress upon rupture of first the parasitophorous vacuole membrane (PVM), then poration and rupture of the RBC membrane (RBCM). Egress is protease-dependent 1 , but none of the effector molecules that mediate membrane rupture have been identified and it is unknown how sequential rupture of the two membranes is controlled. Minutes before egress, the parasite serine protease SUB1 is discharged into the parasitophorous vacuole2-6 where it cleaves multiple substrates2,5,7-9 including SERA6, a putative cysteine protease10-12. Here, we show that Plasmodium falciparum parasites lacking SUB1 undergo none of the morphological transformations that precede egress and fail to rupture the PVM. In contrast, PVM rupture and RBCM poration occur normally in SERA6-null parasites but RBCM rupture does not occur. Complementation studies show that SERA6 is an enzyme that requires processing by SUB1 to function. RBCM rupture is associated with SERA6-dependent proteolytic cleavage within the actin-binding domain of the major RBC cytoskeletal protein ß-spectrin. We conclude that SUB1 and SERA6 play distinct, essential roles in a coordinated proteolytic cascade that enables sequential rupture of the two bounding membranes and culminates in RBCM disruption through rapid, precise, SERA6-mediated disassembly of the RBC cytoskeleton.

Diagnostic challenges of prolonged post-treatment clearance of Plasmodium nucleic acids in a pre-transplant autosplenectomized patient with sickle cell disease.

BACKGROUND: Autosplenectomy, as a result of sickle cell disease, is an important risk factor for severe malaria. While molecular methods are helpful in providing rapid and accurate infection detection and species identification, the effect of hyposplenism on result interpretation during the course of infection should be carefully considered. CASE PRESENTATION: A 32-year old autosplenectomized Nigerian male with severe sickle cell disease was referred to the National Institutes of Health for allogenic hematopoietic stem cell transplant. Despite testing negative for malaria by both smear and PCR 2 weeks after arrival in the USA, the patient developed fever and diffuse bilateral lower rib cage and upper abdominal pain 2 weeks later and subsequently tested positive for Plasmodium falciparum. Parasitaemia was tracked over time by microscopy and nucleic acid tests to evaluate the therapeutic response in the setting of hyposplenism. The patient showed prompt resolution of patent infection by microscopy but remained positive by molecular methods for > 30 days after treatment initiation. CONCLUSION: While molecular testing can provide sensitive Plasmodium nucleic acid detection, the persistence of Plasmodium nucleic acids following adequate treatment in functionally asplenic patients can lead to a diagnostic dilemma. In such patients, clinical response and peripheral blood smears should guide patient management following treatment. Nonetheless, in pre-transplant patients at high-risk for pre-existing Plasmodium infections, highly sensitive molecular assays can be useful to rule out infection prior to transplantation.

Small molecule-based inhibition of MEK1/2 proteins dampens inflammatory responses to malaria, reduces parasite load, and mitigates pathogenic outcomes.

Malaria infections cause several systemic and severe single- or multi-organ pathologies, killing hundreds of thousands of people annually. Considering the existing widespread resistance of malaria parasites to anti-parasitic drugs and their high propensity to develop drug resistance, alternative strategies are required to manage malaria infections. Because malaria is a host immune response-driven disease, one approach is based on gaining a detailed understanding of the molecular and cellular processes that modulate malaria-induced innate and adaptive immune responses. Here, using a mouse cerebral malaria model and small-molecule inhibitors, we demonstrate that inhibiting MEK1/2, the upstream kinases of ERK1/2 signaling, alters multifactorial components of the innate and adaptive immune responses, controls parasitemia, and blocks pathogenesis. Specifically, MEK1/2 inhibitor treatment up-regulated B1 cell expansion, IgM production, phagocytic receptor expression, and phagocytic activity, enhancing parasite clearance by macrophages and neutrophils. Further, the MEK1/2 inhibitor treatment down-regulated pathogenic pro-inflammatory and helper T cell 1 (Th1) responses and up-regulated beneficial anti-inflammatory cytokine responses and Th2 responses. These inhibitor effects resulted in reduced granzyme B expression by T cells, chemokine and intracellular cell adhesion molecule 1 (ICAM-1) expression in the brain, and chemokine receptor expression by both myeloid and T cells. These bimodal effects of the MEK1/2 inhibitor treatment on immune responses contributed to decreased parasite biomass, organ inflammation, and immune cell recruitment, preventing tissue damage and death. In summary, we have identified several previously unrecognized immune regulatory processes through which a MEK1/2 inhibitor approach controls malaria parasitemia and mitigates pathogenic effects on host organs.

Plasmodium falciparum OTU-like cysteine protease (PfOTU) is essential for apicoplast homeostasis and associates with noncanonical role of Atg8.

The metabolic pathways associated with the mitochondrion and the apicoplast in Plasmodium, 2 parasite organelles of prokaryotic origin, are considered as suitable drug targets. In the present study, we have identified functional role of a novel ovarian tumour unit (OTU) domain-containing cysteine protease of Plasmodium falciparum (PfOTU). A C-terminal regulatable fluorescent affinity tag on native protein was utilised for its localization and functional characterization. Detailed studies showed vesicular localization of PfOTU and its association with the apicoplast. Degradation-tag mediated knockdown of PfOTU resulted in abnormal apicoplast development and blocked development of parasites beyond early-schizont stages in subsequent cell cycle; downregulation of PfOTU hindered apicoplast protein import. Further, the isoprenoid precursor-mediated parasite growth-rescue experiments confirmed that PfOTU knockdown specifically effect development of functional apicoplast. We also provide evidence for a possible biological function of PfOTU in membrane deconjugation of Atg8, which may be linked with the apicoplast protein import. Overall, our results show that the PfOTU is involved in apicoplast homeostasis and associates with the noncanonical function of Atg8 in maintenance of parasite apicoplast.