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BACKGROUNDPrediction of adverse outcomes in cerebral malaria (CM) is difficult. We hypothesized that cell-free DNA (cfDNA) levels would facilitate identification of severe and potentially fatal CM cases.METHODSIn this retrospective study, plasma from Malawian children with CM (n = 134), uncomplicated malaria (UM, n = 77), and healthy controls (HC, n = 60) was assayed for cfDNA using a fluorescence assay. Host and parasite cfDNA was measured by quantitative PCR. Immune markers were determined by ELISA, Luminex, or cytometric bead array.RESULTSTotal cfDNA increased with malaria severity (HC versus UM, P < 0.001; HC versus CM, P < 0.0001; UM versus CM, P < 0.0001), was elevated in retinopathy-positive (Ret+) CM relative to Ret- CM (7.66 versus 5.47 ng/μL, P = 0.027), and differentiated Ret+ fatal cases from survivors (AUC 0.779; P < 0.001). cfDNA levels in patients with non-malarial febrile illness (NMF, P = 0.25) and non-malarial coma (NMC, P = 0.99) were comparable with UM. Host DNA, rather than parasite DNA, was the major cfDNA contributor (UM, 268 versus 67 pg/μL; CM, 2824 versus 463 pg/μL). Host and parasite cfDNA distinguished CM by retinopathy (host, AUC 0.715, P = 0.0001; parasite, AUC 0.745, P = 0.0001), but only host cfDNA distinguished fatal cases (AUC 0.715, P = 0.0001). Total cfDNA correlated with neutrophil markers IL-8 (rs = 0.433, P < 0.0001) and myeloperoxidase (rs = 0.683, P < 0.0001).CONCLUSIONQuantifying plasma cfDNA is a simple assay useful in identifying children at risk for fatal outcome and has promise as a point-of-care assay. Elevated cfDNA suggests a link with host inflammatory pathways in fatal CM.FUNDINGNIH NCATS (AK), Burroughs-Wellcome (AK), and National Health and Medical Research Council of Australia (SJR).

Despite an unprecedented 2 decades of success, the combat against malaria - the mosquito-transmitted disease caused by Plasmodium parasites - is no longer progressing. Efforts toward eradication are threatened by the lack of an effective vaccine and a rise in antiparasite drug resistance. Alternative approaches are urgently needed. Repurposing of available, approved drugs with distinct modes of action are being considered as viable and immediate adjuncts to standard antimicrobial treatment. Such strategies may be well suited to the obligatory and clinically silent first phase of Plasmodium infection, where massive parasite replication occurs within hepatocytes in the liver. Here, we report that the widely used antidiabetic drug, metformin, impairs parasite liver stage development of both rodent-infecting Plasmodium berghei and human-infecting P. falciparum parasites. Prophylactic treatment with metformin curtails parasite intracellular growth in vitro. An additional effect was observed in mice with a decrease in the numbers of infected hepatocytes. Moreover, metformin provided in combination with conventional liver- or blood-acting antimalarial drugs further reduced the total burden of P. berghei infection and substantially lessened disease severity in mice. Together, our findings indicate that repurposing of metformin in a prophylactic regimen could be considered for malaria chemoprevention.

The lifestyle of intracellular pathogens, such as malaria parasites, is intimately connected to that of their host, primarily for nutrient supply. Nutrients act not only as primary sources of energy but also as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions. Canonical nutrient-sensing pathways are presumed to be absent from the causative agent of malaria, Plasmodium, thus raising the question of whether these parasites can sense and cope with fluctuations in host nutrient levels. Here we show that Plasmodium blood-stage parasites actively respond to host dietary calorie alterations through rearrangement of their transcriptome accompanied by substantial adjustment of their multiplication rate. A kinome analysis combined with chemical and genetic approaches identified KIN as a critical regulator that mediates sensing of nutrients and controls a transcriptional response to the host nutritional status. KIN shares homology with SNF1/AMPKα, and yeast complementation studies suggest that it is part of a functionally conserved cellular energy-sensing pathway. Overall, these findings reveal a key parasite nutrient-sensing mechanism that is critical for modulating parasite replication and virulence.

Manipulation of the master regulator of energy homeostasis AMP-activated protein kinase (AMPK) activity is a strategy used by many intracellular pathogens for successful replication. Infection by most pathogens leads to an activation of host AMPK activity due to the energetic demands placed on the infected cell. Here, we demonstrate that the opposite is observed in cells infected with rodent malaria parasites. Indeed, AMPK activity upon the infection of hepatic cells is suppressed and dispensable for successful infection. By contrast, an overactive AMPK is deleterious to intracellular growth and replication of different Plasmodium spp., including the human malaria parasite, P. falciparum. The negative impact of host AMPK activity on infection was further confirmed in mice under conditions that activate its function. Overall, this work establishes the role of host AMPK signaling as a suppressive pathway of Plasmodium hepatic infection and as a potential target for host-based antimalarial interventions., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Apicomplexans are obligate intracellular parasites that invade host cells by an active process leading to the formation of a non-fusogenic parasitophorous vacuole (PV) where the parasite replicates within the host cell. The rhomboid family of proteases cleaves substrates within their transmembrane domains and has been implicated in the invasion process. Although its exact function is unknown, Plasmodium ROM1 is hypothesized to play a role during invasion based on its microneme localization and its ability to cleave essential invasion adhesins. Using the rodent malaria model, Plasmodium yoelii, we carried out detailed quantitative analysis of pyrom1 deficient parasites during the Plasmodium lifecycle. Pyrom1(-) parasites are attenuated during erythrocytic and hepatic stages but progress normally through the mosquito vector with normal counts of oocyst and salivary gland sporozoites. Pyrom1 steady state mRNA levels are upregulated 20-fold in salivary gland sporozoites compared to blood stages. We show that pyrom1(-) sporozoites are capable of gliding motility and traversing host cells normally. Wildtype and pyrom1(-) sporozoites do not differ in the rate of entry into Hepa1-6 hepatocytes. Within the first twelve hours of hepatic development, however, only 50% pyrom1(-) parasites have developed into exoerythrocytic forms. Immunofluorescence microscopy using the PVM marker UIS4 and transmission electron microscopy reveal that the PV of a significant fraction of pyrom1(-) parasites are morphologically aberrant shortly after invasion. We propose a novel function for PyROM1 as a protease that promotes proper PV modification to allow parasite development and replication in a suitable environment within the mammalian host.