Toward forward genetic screens in malaria-causing parasites using the piggyBac transposon

The ability to analyze gene function in malaria-causing Plasmodium parasites has received a boost with a recent paper in BMC Genomics that describes a genome-wide mutagenesis system in the rodent malaria species Plasmodium berghei using the transposon piggyBac. This advance holds promise for identifying and validating new targets for intervention against malaria. But further improvements are still needed for the full power of genome-wide molecular genetic screens to be utilized in this organism.

Host cell remodelling and protein trafficking

Inside their respective vertebrate hosts, Plasmodium spp spend most of their life residing within hepatocytes and erythrocytes, with large-scale infection of the latter responsible for the clinical symptoms associated with malaria. These parasites extensively remodel these host cells for a variety of purposes relating to both pathogenesis and maintaining growth. Remodelling of the erythrocytic stage has been most intensively studied in P. falciparum and is the subject of this chapter. To help remodel their hosts these parasites export hundreds of proteins into the erythrocytic compartment. This principally alters the architecture of the erythrocyte, rendering the host membrane more permeable to solutes and nutrients, and also increasing the rigidity and adhesiveness of the infected erythrocyte. Moreover, because erythrocytes lack a secretory apparatus, the parasite must also export many additional proteins to help traffic other proteins to their correct destination within the host cell. The functions of some of these exported proteins will be discussed as will recent progress that has been made in unravelling how exported proteins gain access to the host compartment.

CD8+ T cells from a novel T cell receptor transgenic mouse induce liver-stage immunity that can be boosted by blood-stage infection in rodent malaria

To follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections.

PTEX is an essential nexus for protein export in malaria parasites

During the blood stages of malaria, several hundred parasite-encoded proteins are exported beyond the double-membrane barrier that separates the parasite from the host cell cytosol. These proteins have a variety of roles that are essential to virulence or parasite growth. There is keen interest in understanding how proteins are exported and whether common machineries are involved in trafficking the different classes of exported proteins. One potential trafficking machine is a protein complex known as the Plasmodium translocon of exported proteins (PTEX). Although PTEX has been linked to the export of one class of exported proteins, there has been no direct evidence for its role and scope in protein translocation. Here we show, through the generation of two parasite lines defective for essential PTEX components (HSP101 or PTEX150), and analysis of a line lacking the non-essential component TRX2 (ref. 12), greatly reduced trafficking of all classes of exported proteins beyond the double membrane barrier enveloping the parasite. This includes proteins containing the PEXEL motif (RxLxE/Q/D) and PEXEL-negative exported proteins (PNEPs). Moreover, the export of proteins destined for expression on the infected erythrocyte surface, including the major virulence factor PfEMP1 in Plasmodium falciparum, was significantly reduced in PTEX knockdown parasites. PTEX function was also essential for blood-stage growth, because even a modest knockdown of PTEX components had a strong effect on the parasite's capacity to complete the erythrocytic cycle both in vitro and in vivo. Hence, as the only known nexus for protein export in Plasmodium parasites, and an essential enzymic machine, PTEX is a prime drug target.

An amoeboid algorithm for solving linear transportation problem

Transportation Problem (TP) is one of the basic operational research problems, which plays an important role in many practical applications. In this paper, a bio-inspired mathematical model is proposed to handle the Linear Transportation Problem (LTP) in directed networks by modifying the original amoeba model Physarum Solver. Several examples are used to prove that the provided model can effectively solve Balanced Transportation Problem (BTP), Unbalanced Transportation Problem (UTP), especially the Generalized Transportation Problem (GTP), in a nondiscrete way. © 2013 Elsevier B.V. All rights reserved.

Advances in molecular genetic systems in malaria

Robust tools for analysing gene function in Plasmodium parasites, which are the causative agents of malaria, are being developed at an accelerating rate. Two decades after genetic technologies for use in Plasmodium spp. were first described, a range of genetic tools are now available. These include conditional systems that can regulate gene expression at the genome, transcriptional or protein level, as well as more sophisticated tools for gene editing that use piggyBac transposases, integrases, zinc-finger nucleases or the CRISPR-Cas9 system. In this Review, we discuss the molecular genetic systems that are currently available for use in Plasmodium falciparum and Plasmodium berghei, and evaluate the advantages and limitations of these tools. We examine the insights that have been gained into the function of genes that are important during the blood stages of the parasites, which may help to guide the development and improvement of drug therapies and vaccines.

Novel endoperoxide antimalarials: synthesis, heme binding, and antimalarial activity

We report the synthesis of a series of novel epoxy endoperoxide compounds that can be prepared in high yields in one to three steps from simple starting materials. Some of these compounds inhibit the growth of Plasmodium falciparum in vitro. Structure-activity studies indicate that an endoperoxide ring bisubstituted with saturated cyclic moieties is the pharmacophore. To study the molecular basis of the action of these novel antimalarial compounds, we examined their ability to interact with oxidized and reduced forms of heme. Some of the compounds interact with oxidized heme in a fashion similar to chloroquine and other 4-aminoquinolines, while some of the compounds interact with reduced heme. However, the level of antimalarial potency is not well correlated with these activities, suggesting that some of the endoperoxides may exert their antimalarial activities by a novel mechanism of action.

A new model to imitate the foraging behavior of Physarum polycephalum on a nutrient-poor substrate

Researches on Physarum polycephalum show that methods inspired by the primitive unicellular organism can construct an efficient network and solve some complex problems in graph theory. Current models simulating the intelligent behavior of Physarum are mainly based on Hagen-Poiseuille Law and Kirchhoff Law, reaction-diffusion, Cellular Automaton and multi-agent approach. In this paper, based on an assumption that the plasmodium of Physarum forages for food along the gradient of chemo-attractants on a nutrient-poor substrate, a new model is proposed to imitate its intelligent foraging behavior. The key point of the model is that the growth of Physarum is determined by the simple particle concentration field relating the distance to food source and the shape of food source on a nutrient-poor substrate. To verify this model, numerical experiments are conducted according to Adamatzky[U+05F3]s experiment. Results in spanning tree construction by this model are almost the same as those of Physarum and Oregonator model. The proposed model can also imitate Physarum to avoid repellents. Furthermore, the Euclidean Spanning tree built by this model is similar to its corresponding Minimal Euclidean Spanning tree.

Effect of lactoferrin protein on red blood cells and macrophages: mechanism of parasite-host interaction

BACKGROUND: Lactoferrin is a natural multifunctional protein known to have antitumor, antimicrobial, and anti-inflammatory activity. Apart from its antimicrobial effects, lactoferrin is known to boost the immune response by enhancing antioxidants. Lactoferrin exists in various forms depending on its iron saturation. The present study was done to observe the effect of lactoferrin, isolated from bovine and buffalo colostrum, on red blood cells (RBCs) and macrophages (human monocytic cell line-derived macrophages THP1 cells). METHODS: Lactoferrin obtained from both species and in different iron saturation forms were used in the present study, and treatment of host cells were given with different forms of lactoferrin at different concentrations. These treated host cells were used for various studies, including morphometric analysis, viability by MTT assay, survivin gene expression, production of reactive oxygen species, phagocytic properties, invasion assay, and Toll-like receptor-4, Toll-like receptor-9, and MDR1 expression, to investigate the interaction between lactoferrin and host cells and the possible mechanism of action with regard to parasitic infections. RESULTS: The mechanism of interaction between host cells and lactoferrin have shown various aspects of gene expression and cellular activity depending on the degree of iron saturation of lactoferrin. A significant increase (P<0.05) in production of reactive oxygen species, phagocytic activity, and Toll-like receptor expression was observed in host cells incubated with iron-saturated lactoferrin when compared with an untreated control group. However, there was no significant (P>0.05) change in percentage viability in the different groups of host cells treated, and no downregulation of survivin gene expression was found at 48 hours post-incubation. Upregulation of the Toll-like receptor and downregulation of the P-gp gene confirmed the immunomodulatory potential of lactoferrin protein. CONCLUSION: The present study details the interaction between lactoferrin and parasite host cells, ie, RBCs and macrophages, using various cellular processes and expression studies. The study reveals the possible mechanism of action against various intracellular pathogens such as Toxoplasma, Plasmodium, Leishmania, Trypanosoma, and Mycobacterium. The presence of iron in lactoferrin plays an important role in enhancing the various activities taking place inside these cells. This work provides a lot of information about targeting lactoferrin against many parasitic infections which can rule out the exact pathways for inhibition of diseases caused by intracellular microbes mainly targeting RBCs and macrophages for their survival. Therefore, this initial study can serve as a baseline for further evaluation of the mechanism of action of lactoferrin against parasitic diseases, which is not fully understood to date.

The PTEX component EXP2 is critical for establishing a patent malaria infection in mice

Export of most malaria proteins into the erythrocyte cytosol requires the Plasmodium Translocon of Exported proteins (PTEX) and a cleavable Plasmodium Export Element (PEXEL). In contrast, the contribution of PTEX in the liver stages and export of liver stage proteins is unknown. Here, using the FLP/FRT conditional mutatagenesis system, we generate transgenic P. berghei parasites deficient in EXP2, the putative pore-forming component of PTEX. Our data reveal that EXP2 is important for parasite growth in the liver and critical for parasite transition to the blood, with parasites impaired in their ability to generate a patent blood-stage infection. Surprisingly, whilst parasites expressing a functional PTEX machinery can efficiently export a PEXEL-bearing GFP reporter into the erythrocyte cytosol during a blood stage infection, this same reporter aggregates in large accumulations within the confines of the parasitophorous vacuole membrane during hepatocyte growth. Notably HSP101, the putative molecular motor of PTEX, could not be detected during the early liver stages of infection, which may explain why direct protein translocation of this soluble PEXEL-bearing reporter or indeed native PEXEL proteins into the hepatocyte cytosol has not been observed. This suggests that PTEX function may not be conserved between the blood and liver stages of malaria infection. This article is protected by copyright. All rights reserved.

Migration strategy and pathogen risk: non-breeding distribution drives malaria prevalence in migratory waders

Pathogen exposure has been suggested as one of the factors shaping the myriad of migration strategies observed in nature. Two hypotheses relate migration strategies to pathogen infection: the 'avoiding the tropics hypothesis' predicts that pathogen prevalence and transmission increase with decreasing non-breeding (wintering) latitude, while the "habitat selection hypothesis" predicts lower pathogen prevalence in marine than in freshwater habitats. We tested these scarcely investigated hypotheses by screening wintering and resident wading shorebirds (Charadriiformes) for avian malaria blood parasites (Plasmodium and Haemoproteus spp.) along a latitudinal gradient in Australia. We sequenced infections to determine if wintering migrants share malaria parasites with local shorebird residents, and we combined prevalence results with published data in a global comparative analysis. Avian malaria prevalence in Australian waders was 3.56% and some parasite lineages were shared between wintering migrants and residents, suggesting active transmission at wintering sites. In the global dataset, avian malaria prevalence was highest during winter and increased with decreasing wintering latitude, after controlling for phylogeny. The latitudinal gradient was stronger for waders that use marine and freshwater habitats (marine + freshwater) than for marine-restricted species. Marine + freshwater wader species also showed higher overall avian malaria parasite prevalence than marine-restricted species. By combining datasets in a global comparative analysis, we provide empirical evidence that migratory waders avoiding the tropics during the non-breeding season experience a decreased risk of malaria parasite infection. We also find global support for the hypothesis that marine-restricted shorebirds experience lower parasite pressures than shorebirds that also use freshwater habitats. Our study indicates that pathogen transmission may be an important driver of site selection for non-breeding migrants, a finding that contributes new knowledge to our understanding of how migration strategies evolve.

Host cell remodelling in malaria parasites: a new pool of potential drug targets

When in their human hosts, malaria parasites spend most of their time housed within vacuoles inside erythrocytes and hepatocytes. The parasites extensively modify their host cells to obtain nutrients, prevent host cell breakdown and avoid the immune system. To perform these modifications, malaria parasites export hundreds of effector proteins into their host cells and this process is best understood in the most lethal species to infect humans, Plasmodium falciparum. The effector proteins are synthesized within the parasite and following a proteolytic cleavage event in the endoplasmic reticulum and sorting of mature proteins into the correct vesicular trafficking pathway, they are transported to the parasite surface and released into the vacuole. The effector proteins are then unfolded before extrusion across the vacuole membrane by a unique translocon complex called Plasmodium translocon of exported proteins. After gaining access to the erythrocyte cytoplasm many effector proteins continue their journey to the erythrocyte surface by utilising various membranous structures established by the parasite. This complex trafficking pathway and a large number of the effector proteins are unique to Plasmodium parasites. This pathway could, therefore, be developed as new drug targets given that protein export and the functional role of these proteins are essential for parasite survival. This review explores known and potential drug targetable steps in the protein export pathway and strategies for discovering novel drug targets.

Ecology of urban malaria vectors in Niamey, Republic of Niger

BACKGROUND: Urbanization in African cities has major impact on malaria risk. Niamey, the capital of the Republic of Niger, is situated in the West African Sahel zone. The short rainy season and human activities linked with the Niger River influence mosquito abundance. This study aimed at deciphering the factors of distribution of urban malaria vectors in Niamey.

METHODS: The distribution of mosquito aquatic stages was investigated monthly from December 2002 to November 2003, at up to 84 breeding sites, throughout Niamey. An exploratory analysis of association between mosquito abundance and environmental factors was performed by a Principal Component Analysis and confirmed by Kruskall-Wallis non-parametric test. To assess the relative importance of significant factors, models were built for Anopheles and Culicinae. In a second capture session, adult mosquitoes were collected weekly with pyrethrum sprays and CDC light-traps from June 2008 to June 2009 in two differentiated urban areas chosen after the study's first step. Members of the Anopheles gambiae complex were genotyped and Anopheles females were tested for the presence of Plasmodium falciparum circumsporozoite antigens using ELISA.

RESULTS: In 2003, 29 % of 8420 mosquitoes collected as aquatic stages were Anopheles. They were significantly more likely to be found upstream, relatively close to the river and highly productive in ponds. These factors remained significant in regression and generalized linear models. The Culicinae were found significantly more likely close to the river, and in the main temporary affluent stream. In 2009, Anopheles specimens, including Anopheles gambiae s.l. (95 %), but also Anopheles funestus (0.6 %) accounted for 18 % of the adult mosquito fauna, with a large difference between the two sampled zones. Three members of the An. gambiae complex were found: Anopheles arabiensis, Anopheles coluzzii, and An. gambiae. Nineteen (1.3 %) out of 1467 females tested for P. falciparum antigen were found positive.

CONCLUSION: The study provides valuable update knowledge on malaria vector ecology and distribution in Niamey. The identification of spatial and environmental risk factors could pave the way to larval source management strategy and allow malaria vector control to focus on key zones for the benefit of the community.

Dissecting the function of essential rhoptry genes of the malaria parasite

This thesis explored the role of essential Rhoptry-proteins (RAPs) in parasites that cause malaria. Utilising genetic engineering approaches, this work provides the first formal proof that RAPs are not involved in the invasion of host erythrocytes and instead presents evidence supporting a post-invasion role. This changes the existing notion of RAPs as potential vaccine-candidates to being potential drug-targets.