Plasmodium co-infection protects against chikungunya virus-induced pathologies.

Co-infection with Plasmodium and chikungunya virus (CHIKV) has been reported in humans, but the impact of co-infection on pathogenesis remains unclear. Here, we show that prior exposure to Plasmodium suppresses CHIKV-associated pathologies in mice. Mechanistically, Plasmodium infection induces IFNγ, which reduces viraemia of a subsequent CHIKV infection and suppresses tissue viral load and joint inflammation. Conversely, concomitant infection with both pathogens limits the peak of joint inflammation with no effect on CHIKV viraemia. Reduced peak joint inflammation is regulated by elevated apoptosis of CD4+ T-cells in the lymph nodes and disrupted CXCR3-mediated CD4+ T-cell migration that abolishes their infiltration into the joints. Virus clearance from tissues is delayed in both infection scenarios, and is associated with a disruption of B cell affinity-maturation in the spleen that reduces CHIKV-neutralizing antibody production.

The effect of dietary antioxidant supplementation in a vertebrate host on the infection dynamics and transmission of avian malaria to the vector.

Host susceptibility to parasites is likely to be influenced by intrinsic factors, such as host oxidative status determined by the balance between pro-oxidant production and antioxidant defences. As a result, host oxidative status acts as an environmental factor for parasites and may constrain parasite development. We evaluated the role of host oxidative status on infection dynamics of an avian malarial parasite by providing canaries (Serinus canaria) with an antioxidant supplementation composed of vitamin E (a lipophilic antioxidant) and olive oil, a source of monounsaturated fatty acids. Another group received a standard, non-supplemented food. Half of the birds in each group where then infected with the haemosporidian parasite, Plasmodium relictum. We monitored the parasitaemia, haematocrit level, and red cell membrane resistance, as well as the transmission success of the parasite to its mosquito vector, Culex pipiens. During the acute phase, the negative effect of the infection was more severe in the supplemented group, as shown by a lower haematocrit level. Parasitaemia was lower in the supplemented group during the chronic phase only. Mosquitoes fed on supplemented hosts were more often infected than mosquitoes fed on the control group. These results suggest that dietary antioxidant supplementation conferred protection against Plasmodium in the long term, at the expense of a short-term negative effect. Malaria parasites may take advantage of antioxidants, as shown by the increased transmission rate in the supplemented group. Overall, our results suggest an important role of oxidative status in infection outcome and parasite transmission.

Discovery of Plasmodium (M)TRAP-Aldolase Interaction Stabilizers Interfering with Sporozoite Motility and Invasion.

As obligate, intracellular parasites, Plasmodium spp. rely on invasion of host cells in order to replicate and continue their life cycle. The parasite needs to traverse the dermis and endothelium of blood vessels, invade hepatocytes and red blood cells, traverse the mosquito midgut, and enter the salivary glands to continue the cycle of infection and transmission. To traverse and invade cells, the parasite employs an actomyosin motor at the core of a larger invasion machinery complex known as the glideosome. The complex is comprised of multiple protein-protein interactions linking the motor to the internal cytoskeletal network of the parasite and to the extracellular adhesins, which directly contact the host tissue or cell surface. One key interaction is between the cytoplasmic tails of the thrombospondin related anonymous protein (TRAP) and aldolase, a bridging protein to the motor. Here, we present results from screening the Medicines for Malaria Venture (MMV) library of 400 compounds against this key protein-protein interaction. Using a surface plasmon resonance screen, we have identified several compounds that modulate the dynamics of the interaction between TRAP and aldolase. These compounds have been validated in vitro by studying their effects on sporozoite gliding motility and hepatocyte invasion. One of the MMV compounds identified reduced invasion levels by 89% at the lowest concentration tested (16 µM) and severely inhibited gliding at even lower concentrations (5 µM). By targeting protein-protein interactions, we investigated an under-explored area of parasite biology and general drug development, to identify potential antimalarial lead compounds.

Malaria was a weak selective force in ancient Europeans.

Malaria, caused by Plasmodium parasites, is thought to be one of the strongest selective forces that has shaped the genome of modern humans and was endemic in Europe until recent times. Due to its eradication around mid-twentieth century, the potential selective history of malaria in European populations is largely unknown. Here, we screen 224 ancient European genomes from the Upper Palaeolithic to the post-Roman period for 22 malaria-resistant alleles in twelve genes described in the literature. None of the most specific mutations for malaria resistance, like those at G6PD, HBB or Duffy blood group, have been detected among the available samples, while many other malaria-resistant alleles existed well before the advent of agriculture. We detected statistically significant differences between ancient and modern populations for the ATP2B4, FCGR2B and ABO genes and we found evidence of selection at IL-10 and ATP2B4 genes. However it is unclear whether malaria is the causative agent, because these genes are also involved in other immunological challenges. These results suggest that the selective force represented by malaria was relatively weak in Europe, a fact that could be associated to a recent historical introduction of the severe malaria pathogen.

Ex Vivo Maturation Assay for Testing Antimalarial Sensitivity of Rodent Malaria Parasites.

Ex vivo assay systems provide a powerful approach to studying human malaria parasite biology and to testing antimalarials. For rodent malaria parasites, short-term in vitro culture and ex vivo antimalarial susceptibility assays are relatively cumbersome, relying on in vivo passage for synchronization, since ring-stage parasites are an essential starting material. Here, we describe a new approach based on the enrichment of ring-stage Plasmodium berghei, P. yoelii, and P. vinckei vinckei using a single-step Percoll gradient. Importantly, we demonstrate that the enriched ring-stage parasites develop synchronously regardless of the parasite strain or species used. Using a flow cytometry assay with Hoechst and ethidium or MitoTracker dye, we show that parasite development is easily and rapidly monitored. Finally, we demonstrate that this approach can be used to screen antimalarial drugs.

The Role of Oxidative Stress in the Longevity and Insecticide Resistance Phenotype of the Major Malaria Vectors Anopheles arabiensis and Anopheles funestus.

Oxidative stress plays numerous biological roles, both functional and pathological. The role of oxidative stress in various epidemiologically relevant biological traits in Anopheles mosquitoes is not well established. In this study, the effects of oxidative stress on the longevity and insecticide resistance phenotype in the major malaria vector species An. arabiensis and An. funestus were examined. Responses to dietary copper sulphate and hydrogen peroxide were used as proxies for the oxidative stress phenotype by determining the effect of copper on longevity and hydrogen peroxide lethal dose. Glutathione peroxidase and catalase activities were determined colorimetrically. Oxidative burden was quantified as protein carbonyl content. Changes in insecticide resistance phenotype were monitored by WHO bioassay. Insecticide resistant individuals showed an increased capacity for coping with oxidative stress, mediated by increased glutathione peroxidase and catalase activity. This effect was observed in both species, as well as in laboratory strains and F1 individuals derived from wild-caught An. funestus mothers. Phenotypic capacity for coping with oxidative stress was greatest in strains with elevated Cytochrome P450 activity. Synergism of oxidative stress defence enzymes by dietary supplementation with haematin, 3-Amino-1, 2, 4-triazole and Sodium diethyldithiocarbamate significantly increased pyrethroid-induced mortality in An. arabiensis and An. funestus. It is therefore concluded that defence against oxidative stress underlies the augmentation of the insecticide resistance phenotype associated with multiple blood-feeding. This is because multiple blood-feeding ultimately leads to a reduction of oxidative stress in insecticide resistant females, and also reduces the oxidative burden induced by DDT and pyrethroids, by inducing increased glutathione peroxidase activity. This study highlights the importance of oxidative stress in the longevity and insecticide resistance phenotype in malaria vectors.

Testing in mice the hypothesis that melanin is protective in malaria infections.

Malaria has had the largest impact of any infectious disease on shaping the human genome, exerting enormous selective pressure on genes that improve survival in severe malaria infections. Modern humans originated in Africa and lost skin melanization as they migrated to temperate regions of the globe. Although it is well documented that loss of melanization improved cutaneous Vitamin D synthesis, melanin plays an evolutionary ancient role in insect immunity to malaria and in some instances melanin has been implicated to play an immunoregulatory role in vertebrates. Thus, we tested the hypothesis that melanization may be protective in malaria infections using mouse models. Congenic C57BL/6 mice that differed only in the gene encoding tyrosinase, a key enzyme in the synthesis of melanin, showed no difference in the clinical course of infection by Plasmodium yoelii 17XL, that causes severe anemia, Plasmodium berghei ANKA, that causes severe cerebral malaria or Plasmodium chabaudi AS that causes uncomplicated chronic disease. Moreover, neither genetic deficiencies in vitamin D synthesis nor vitamin D supplementation had an effect on survival in cerebral malaria. Taken together, these results indicate that neither melanin nor vitamin D production improve survival in severe malaria.

Imaging of Plasmodium liver stages to drive next-generation antimalarial drug discovery.

Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.

Malaria infection and human evolution.

During the evolution of the genus Homo, with regard to the species habilis, erectus and sapiens, malaria has played a key biological role in influencing human development. The plasmodia causing malaria have evolved in two ways, in biological and phylogenetic terms: Plasmodium vivax, Plasmodium malariae and Plasmodium ovale appear to have either coevolved with human mankind, or encountered human species during the most ancient phases of Homo evolution; on the other hand, Plasmodium falciparum has been transmitted to humans by monkeys in a more recent period, probably between the end of the Mesolithic and the beginning of the Neolithic age. The authors show both direct and indirect biomolecular evidence of malarial infection, detected in buried subjects, dating to ancient times and brought to light in the course of archaeological excavations in major Mediterranean sites. In this review of the literature the authors present scientific evidence confirming the role of malaria in affecting the evolution of populations in Mediterranean countries. The people living in several different Mediterranean regions, the cradle of western civilization, have been progressively influenced by malaria in the course of the spread of this endemic disease in recent millennia. In addition, populations affected by endemic malaria progressively developed cultural, dietary and behavioural adaptation mechanisms, which contributed to diminish the risk of disease. These habits were probably not fully conscious. Nevertheless it may be thought that both these customs and biological modifications, caused by malarial plasmodia, favoured the emergence of groups of people with greater resistance to malaria. All these factors have diminished the unfavourable demographic impact of the disease, also positively influencing the general development and growth of civilization.

Antibiotics in malaria therapy and their effect on the parasite apicoplast.

The impact of selected antibiotics on combating malaria infections was discovered in the mid of last century. Only recently, studies on their modes of action in malaria parasites have been initiated, prompted by the discovery of a prokaryotic organelle, the apicoplast. This plastid-derived structure, which originates from a secondary endosymbiotic event, possesses important metabolic as well as housekeeping functions, including fatty acid and heme biosynthesis. Due to its indispensability for parasite survival it represents a promising target for the use of antibiotics in malaria therapy. Most antibiotics cause a delayed death phenotype, which manifests in the late onset of antimalarial activity during the second replication cycle of the pathogen. This review will describe the effect of classical antibacterial agents against malaria parasites and the use of some of these compounds in clinical settings. Firstly we discuss the current knowledge about the physiological and morphological effects of antibiotics on the parasite and the apicoplast in particular, with special focus on the delayed death effect. Secondly antimalarial antibiotics are specified and their effects in vitro are compared with available in vivo data and clinical studies. Major precautions and side effects are described.

RXLR-mediated entry of Phytophthora sojae effector Avr1b into soybean cells does not require pathogen-encoded machinery.

Effector proteins secreted by oomycete and fungal pathogens have been inferred to enter host cells, where they interact with host resistance gene products. Using the effector protein Avr1b of Phytophthora sojae, an oomycete pathogen of soybean (Glycine max), we show that a pair of sequence motifs, RXLR and dEER, plus surrounding sequences, are both necessary and sufficient to deliver the protein into plant cells. Particle bombardment experiments demonstrate that these motifs function in the absence of the pathogen, indicating that no additional pathogen-encoded machinery is required for effector protein entry into host cells. Furthermore, fusion of the Avr1b RXLR-dEER domain to green fluorescent protein (GFP) allows GFP to enter soybean root cells autonomously. The conclusion that RXLR and dEER serve to transduce oomycete effectors into host cells indicates that the >370 RXLR-dEER-containing proteins encoded in the genome sequence of P. sojae are candidate effectors. We further show that the RXLR and dEER motifs can be replaced by the closely related erythrocyte targeting signals found in effector proteins of Plasmodium, the protozoan that causes malaria in humans. Mutational analysis of the RXLR motif shows that the required residues are very similar in the motifs of Plasmodium and Phytophthora. Thus, the machinery of the hosts (soybean and human) targeted by the effectors may be very ancient.

Acidocalcisomes in Apicomplexan parasites.

Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to man. They posses an acidic matrix that contains several cations bound to phosphates, mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. The calcium uptake occurs through a Ca2+/H+ counter transporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. In this paper, we review the structural, biochemical and physiological aspects of acidocalcisomes in Apicomplexan parasites and discuss their functional roles in the maintenance of intracellular ion homeostasis.

The disease triangle: pathogens, the environment and society.

The primary means to define any disease is by naming a pathogen or agent that negatively affects the health of the host organism. Another assumed, but often overlooked, determinant of disease is the environment, which includes deleterious physical and social effects on mankind. The disease triangle is a conceptual model that shows the interactions between the environment, the host and an infectious (or abiotic) agent. This model can be used to predict epidemiological outcomes in plant health and public health, both in local and global communities. Here, the Irish potato famine of the mid-nineteenth century is used as an example to show how the disease triangle, originally devised to interpret plant disease outcomes, can be applied to public health. In parallel, malaria is used to discuss the role of the environment in disease transmission and control. In both examples, the disease triangle is used as a tool to discuss parameters that influence socioeconomic outcomes as a result of host-pathogen interactions involving plants and humans.

Target-based drug discovery for malaria, leishmaniasis, and trypanosomiasis.

Advances in combinatorial chemistry, high-throughput screening, and molecular modeling have revolutionized the process of drug discovery in the pharmaceutical industry. Drug discovery efforts for the primary protozoal parasitic diseases of the developing world, malaria, leishmaniasis, and trypanosomiasis, have also begun to employ these techniques. Drug targets in these parasites, exemplified by cysteine proteases and trypanothione reductase, have been purified and used for inhibitor screening. Through this work, small molecules have been identified that inhibit both parasite proteins and the growth of the organisms. This review describes advances that have been made in examining the effects of small molecules on potential parasitic drug targets determined by biochemical and computer-based screening, and also details the activity of such compounds on parasites in vitro and in vivo. Based on these results, it is apparent that modern drug discovery techniques hold promise for the identification of antiparasitic drug candidates.

[Timing niches of 3 species of Plasmodium coexisting in a rodent in Central Africa]. / Niches horaires des trois espèces de Plasmodies coexistant chez un Rongeur de Centrafrique.

Freeze-thawing of blood infected with malaria parasites is a technique which brings about the destruction of all stages except the merozoites and makes possible investigations on the behaviour of these merozoites and the schizogonic rhythm of each species. Merozoites of Plasmodium y. yoelii remain in the blood during the 24 hrs. following inoculation; it is concluded that their penetration in the erythrocytes occurs gradually during this time. Synchronism is poor. Merozoites of P. vinckei petteri penetrate rapidly inside the erythrocytes independently of the time of inoculation. Infection is therefore synchronous and does not follow the circadian rhythm of the host. Penetration of merozoites of P. c. chabaudi is predominant at midnight when rodents are maintained with a normal circadian rhythm (light from 8 am to 8 pm) and predominant at noon when the rhythm of the host is inverted (light from 8 pm to 8 am). Infection is therefore synchronous and follows the host rhythm. The three species of plasmodia coexisting in Thamnomys rutilans from CAR show the same periodicity of 24 hrs. but, because of differences in the biology of the merozoites, they occupy three distinct niches. These notions have great practical implications in chronotherapy, as many data lead to the idea that merozoites are drug resistant.

[Control of malaria--chemotherapy and immunoprophylaxis. 1. Chemotherapy]. / Malariabekämpfung--Chemotherapie und Immunprophylaxe. 1. Mitteilung: Chemotherapie.

A survey on the advances of the fight against malaria is given, and the present world-wide situation of this disease is described including the important role World Health Organization (WHO) is playing in this fight. The chapter on chemotherapy deals with recent progress in this field with special regard to mefloquine and quinghaosu, but other newer compounds are also discussed. The prospects of vaccination against malaria in general are considered, and the results of in vitro and in vivo experiments using antigens from sporozoites, asexual blood stages and gametocytes are reported. Humoral and cellular immunity and their part in the immune response of the infected host are evaluated as well as the progress in the continuous cultivation of the malaria parasites. Finally, the perspectives of the fight against malaria in the years to come are discussed.