The dynamics of interactions between Plasmodium and the mosquito: a study of the infectivity of Plasmodium berghei and Plasmodium gallinaceum,and their transmission by Anopheles stephensi,Anopheles gambiae and Aedes aegypti

Knowledge of parasite-mosquito interactions is essential to develop strategies that will reduce malaria transmission through the mosquito vector. In this study we investigated the development of two model malaria parasites, Plasmodium berghei and Plasmodium gallinaceum, in three mosquito species Anopheles stephensi, Anopheles gambiae and Aedes aegypti. New methods to study gamete production in vivo in combination with GFP-expressing ookinetes were employed to measure the large losses incurred by the parasites during infection of mosquitoes. All three mosquito species transmitted P. gallinaceum; P. berghei was only transmitted by Anopheles spp. Plasmodium gallinaceum initiates gamete production with high efficiency equally in the three mosquito species. By contrast P. berghei is less efficiently activated to produce gametes, and in Ae. aegypti microgamete formation is almost totally suppressed. In all parasite/vector combinations ookinete development is inefficient, 500-100,000-fold losses were encountered. Losses during ookinete-to-oocyst transformation range from fivefold in compatible vector parasite combinations (P. berghei/An. stephensi), through >100-fold in poor vector/parasite combinations (P. gallinaceum/An. stephensi), to complete blockade (>1,500 fold) in others (P. berghei/Ae. aegypti). Plasmodium berghei ookinetes survive poorly in the bloodmeal of Ae. aegypti and are unable to invade the midgut epithelium. Cultured mature ookinetes of P. berghei injected directly into the mosquito haemocoele produced salivary gland sporozoites in An. stephensi, but not in Ae. aegypti, suggesting that further species-specific incompatibilities occur downstream of the midgut epithelium in Ae. aegypti. These results show that in these parasite-mosquito combinations the susceptibility to malarial infection is regulated at multiple steps during the development of the parasites. Understanding these at the molecular level may contribute to the development of rational strategies to reduce the vector competence of malarial vectors.     

Minimum requirements for ookinete to oocyst transformation in Plasmodium

During their passage through a mosquito vector, malaria parasites undergo several developmental transformations including that from a motile zygote, the ookinete, to a sessile oocyst that develops beneath the basal lamina of the midgut epithelium. This transformation process is poorly understood and the oocyst is the least studied of all the stages in the malaria life cycle. We have used an in vitro culture system to monitor morphological features associated with transformation of Plasmodium berghei ookinetes and the role of basal lamina components in this process. We also describe the minimal requirements for transformation and early oocyst development. A defined sequence of events begins with the break-up of the inner surface membrane, specifically along the convex side of the ookinete, where a protrusion occurs. A distinct form, the transforming ookinete or took, has been identified in vitro and also observed in vivo. Contrary to previous suggestions, we have shown that no basal lamina components are required to trigger ookinete to oocyst transformation in vitro. We have demonstrated that transformation does not occur spontaneously; it is initiated in the presence of bicarbonate added to PBS, but it is not mediated by changes in pH alone. Transformation is a two-step process that is not completed unless a range of nutrients are also present. A minimal medium is defined which supports transformation and oocyst growth from 7.8 to 11.4microm by day 5 with 84% viability. We conclude that ookinete transformation is mediated by bicarbonate and occurs in a similar manner to the differentiation of sporozoite to the hepatic stage.     

The role of Plasmodium berghei ookinete proteins in binding to basal lamina components and transformation into oocysts.

The ookinete is a motile form of the malaria parasite that travels from the midgut lumen of the mosquito, invades the epithelial cells and settles beneath the basal lamina. The events surrounding cessation of ookinete motility and its transformation into an oocyst are poorly understood, but interaction between components of the basal lamina and the parasite surface has been implicated. Here we report that interactions occur between basal lamina constituents and ookinete proteins and that these interactions inhibit motility and are likely to be involved in transformation to an oocyst. Plasmodium berghei ookinetes bound weakly to microtitre plate wells coated with fibronectin and much more strongly to wells coated with laminin and collagen IV. A 1:1 mixture of collagen and laminin significantly enhanced binding. Binding increased with time of incubation up to 10 h and different components showed different binding profiles with time. Two parasite molecules were shown to act as ligands for basal lamina components. Western blots demonstrated that the surface molecule Pbs21 bound strongly to laminin but not to collagen IV whereas a 215 kDa molecule (possibly PbCTRP) bound to both laminin and collagen IV. Furthermore up to 90% inhibition of binding of ookinetes to collagen IV/laminin combination occurred if parasites were pre-incubated with anti-Pbs21 monoclonal antibody 13.1. Some transformation of ookinetes to oocysts occurred in wells coated with laminin or laminin/collagen IV combinations but collagen IV alone did not trigger transformation. No binding or transformation occurred in uncoated wells. Our data support the suggestion that ookinete proteins Pbs21 and a 215 kDa protein may have multiple roles including interactions with midgut basal lamina components that cause binding, inhibit motility and trigger transformation.     

A putative kinase-related protein (PKRP) from Plasmodium berghei mediates infection in the midgut and salivary glands of the mosquito

The completion of the Plasmodium (malaria) life cycle in the mosquito requires the parasite to traverse first the midgut and later the salivary gland epithelium. We have identified a putative kinase-related protein (PKRP) that is predicted to be an atypical protein kinase, which is conserved across many species of Plasmodium. The pkrp gene encodes a RNA of about 5300 nucleotides that is expressed as a 90 kDa protein in sporozoites. Targeted disruption of the pkrp gene in Plasmodium berghei, a rodent model of malaria, compromises the ability of parasites to infect different tissues within the mosquito host. Early infection of mosquito midgut is reduced by 58-71%, midgut oocyst production is reduced by 50-90% and those sporozoites that are produced are defective in their ability to invade mosquito salivary glands. Midgut sporozoites are not morphologically different from wild-type parasites by electron microscopy. Some sporozoites that emerged from oocysts were attached to the salivary glands but most were found circulating in the mosquito hemocoel. Our findings indicate that a signalling pathway involving PbPKRP regulates the level of Plasmodium infection in the mosquito midgut and salivary glands.     

Computational prediction of protein interactions related to the invasion of erythrocytes by malarial parasites

Background

The invasion of red blood cells (RBCs) by malarial parasites is an essential step in the life cycle of Plasmodium falciparum. Human-parasite surface protein interactions play a critical role in this process. Although several interactions between human and parasite proteins have been discovered, the mechanism related to invasion remains poorly understood because numerous human-parasite protein interactions have not yet been identified. High-throughput screening experiments are not feasible for malarial parasites due to difficulty in expressing the parasite proteins. Here, we performed computational prediction of the PPIs involved in malaria parasite invasion to elucidate the mechanism by which invasion occurs.

Results

In this study, an expectation maximization algorithm was used to estimate the probabilities of domain-domain interactions (DDIs). Estimates of DDI probabilities were then used to infer PPI probabilities. We found that our prediction performance was better than that based on the information of D. melanogaster alone when information related to the six species was used. Prediction performance was assessed using protein interaction data from S. cerevisiae, indicating that the predicted results were reliable. We then used the estimates of DDI probabilities to infer interactions between 490 parasite and 3,787 human membrane proteins. A small-scale dataset was used to illustrate the usability of our method in predicting interactions between human and parasite proteins. The positive predictive value (PPV) was lower than that observed in S. cerevisiae. We integrated gene expression data to improve prediction accuracy and to reduce false positives. We identified 80 membrane proteins highly expressed in the schizont stage by fast Fourier transform method. Approximately 221 erythrocyte membrane proteins were identified using published mass spectral datasets. A network consisting of 205 interactions was predicted. Results of network analysis suggest that SNARE proteins of parasites and APP of humans may function in the invasion of RBCs by parasites.

Conclusions

We predicted a small-scale PPI network that may be involved in parasite invasion of RBCs by integrating DDI information and expression profiles. Experimental studies should be conducted to validate the predicted interactions. The predicted PPIs help elucidate the mechanism of parasite invasion and provide directions for future experimental investigations.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0393-z) contains supplementary material, which is available to authorized users.     

Ferriprotoporphyrin IX, phospholipids, and the antimalarial actions of quinoline drugs

Two subclasses of quinoline antimalarial drugs are used clinically. Both act on the endolysosomal system of malaria parasites, but in different ways. Treatment with 4-aminoquinoline drugs, such as chloroquine, causes morphologic changes and hemoglobin accumulation in endocytic vesicles. Treatment with quinoline-4-methanol drugs, such as quinine and mefloquine, also causes morphologic changes, but does not cause hemoglobin accumulation. In addition, chloroquine causes undimerized ferriprotoporphyrin IX (ferric heme) to accumulate whereas quinine and mefloquine do not. On the contrary, treatment with quinine or mefloquine prevents and reverses chloroquine-induced accumulation of hemoglobin and undimerized ferriprotoporphyrin IX. This difference is of particular interest since there is convincing evidence that undimerized ferriprotoporphyrin IX in malaria parasites would interact with and serve as a target for chloroquine. According to the ferriprotoporphyrin IX interaction hypothesis, chloroquine would bind to undimerized ferriprotoporphyrin IX, delay its detoxification, cause it to accumulate, and allow it to exert its intrinsic biological toxicities. The ferriprotoporphyrin IX interaction hypothesis appears to explain the antimalarial action of chloroquine, but a drug target in addition to ferriprotoporphyrin IX is suggested by the antimalarial actions of quinine and mefloquine. This article summarizes current knowledge of the role of ferriprotoporphyrin IX in the antimalarial actions of quinoline drugs and evaluates the currently available evidence in support of phospholipids as a second target for quinine, mefloquine and, possibly, the chloroquine-ferriprotoporphyrin IX complex.     

Plasmodium berghei parasite transformed with green fluorescent protein for screening blood schizontocidal agents

High priority has been given to new assays that facilitate and accelerate the development of novel antimalarial compounds. Unlike evaluation of drugs in vitro, in which new approaches have been used to expedite identification of parasites, the conventional in vivo murine assay requires determination of parasitemia by light microscopy, an incompatible technique to test large numbers of drugs. We have investigated the possibility of using an autonomously fluorescent Plasmodium berghei strain, stably transformed with the green fluorescent protein, to rapidly quantify parasite growth by flow cytometry. The major improvement of this method is that P. berghei line transformed with green fluorescent protein parasites can be quickly and specifically detected in a drop of parasite-infected blood without any manipulation of the sample. Our results showed a clear correlation between the numbers of fluorescent cells detected by flow cytometry and conventional parasitemia, including a correspondence in the peaks of parasitemia. The validation of P. berghei line transformed with green fluorescent protein for chemotherapy studies was performed by evaluating its response to conventional antimalarial drugs such as chloroquine, quinine and sodium artesunate. The results of drug-susceptibility assays as determined by flow cytometry were comparable with those obtained by microscopic examination of Giemsa-stained slides. This PbGFP parasite should prove to be a rapid, simple and sensitive tool for the examination of the large number of compounds and conditions involved in the initial stages of drug development.     

Murine malaria. I. Measurement of the mean rate of increase in vivo of Plasmodium berghei

Swiss mice were inoculated intraperitoneally with RBC infected with Plasmodium berghei. The moment a certain parasitemia was reached in each individual mouse was estimated by means of linear interpolation. The relationship between latent period and log inoculum was investigated by means of simple linear regression. The slopes of the latent period per log inoculum curves were significantly different using different donor mice inoculated with serial 10-fold dilution of infected RBC and exsanguinated in the same phase of the infection. It could not be demonstrated that the slopes of the regression lines depended on the stage of the disease of the donor mouse.     

Plasmodium yoelii: novel rhoptry proteins identified within the body of merozoite rhoptries in rodent Plasmodium malaria

The biogenesis, organization and function of the rhoptries are not well understood. Antisera were prepared to synthetic peptides prepared as multiple antigenic peptides (MAPs) obtained from a Plasmodium yoelii merozoite rhoptry proteome analysis. The antisera were used in immunofluorescence and immunoelectron microscopy of schizont-infected erythrocytes. Twenty-seven novel rhoptry proteins representing proteases, metabolic enzymes, secreted proteins and hypothetical proteins, were identified in the body of the rhoptries by immunoelectron microscopy. The merozoite rhoptries contain a heterogeneous mixture of proteins that may initiate host cell invasion and establish intracellular parasite development.     

Wave expansion of CD34 progenitor cells in the spleen in rodent malaria

Defense against malaria depends upon amplification of the spleen structure and function for the clearance of parasitized red blood cells (pRBC). We studied the distribution and amount of CD34⁺ cells in the spleens of mice infected with rodent malaria. We sought to identify these cells in the spleen and determine their relationship to infection. C57BL/6J mice were infected with self-resolving, Plasmodium chabaudi CR, or one of the lethal rodent malaria strains, P. chabaudi AJ and P. berghei ANKA. We then recorded parasitemia, mortality, and the presence of CD34⁺ cells in spleen, as determined by immunohistochemistry and flow cytometry. In the non-lethal strain, the spleen structure was maintained during amplification, but disrupted in lethal models. The abundance of CD34⁺ cells increased in the red pulp on the 4th and 6th days p.i. in all models, and subsided on the 8th day p.i. Faint CD34⁺ staining on the 8th day p.i., was probably due to differentiation of committed cell lineages. In this work, increase of spleen CD34⁺ cells did not correlate with infection control.     

Theories on malarial pigment formation and quinoline action

Haeme metabolism remains a vulnerable problem for the intraerythrocytic Plasmodium which catabolises haemoglobin as a source of amino acids in an acidic, oxygen-rich lysosome-like digestive vacuole. Haeme monomer, capable of generating oxygen radicals, transforms into an inert crystal named malarial pigment or haemozoin by forming unique dimers that then crystalise. Laveran first described pigmented bodies in humans to define a protozoan as the aetiologic agent of malaria. The trail of malaria pigment enabled Ross to implicate the mosquito in the life cycle of Plasmodium. In 1991, Slater and Cerami postulated a unique iron-carboxylate bond between two haemes in haemozoin crystals based on infrared and X-ray spectroscopy data. Additionally, parasite extracts were shown to possess a 'haeme polymerase' enzymatic activity as the process of crystal formation was then termed. Importantly, the quinolines, such as choloroquine, inhibit haemozoin formation. A Plasmodium falciparum derived histidine-rich protein II, which binds haeme and initiates haemozoin formation, is present in the digestive vacuole. Pfhistidine-rich protein II and Pfhistidine-rich protein III are sufficient, but not necessary for haemozoin formation as a laboratory clone lacking both still makes the haeme crystals. The reduvid bug, and the Schistosoma and Haemoproteus genera also make haemozoin. Recently, Bohle and coworkers used X-ray diffraction to document the iron-carboxylate bond in intact desiccated parasites and to show that a Fe1-O41 head to tail haeme dimer is the unit building block of haemozoin. The role of the Plasmodium histidine-rich protein family members, lipids or potential novel proteins in the exact molecular assembly of the large molecular weight haeme crystals in the protein rich digestive vacuole needs to be solved. Accurate experimental determination of the role of haemozoin formation and inhibition as the target of chloroquine is fundamental to determination of the mechanism of quinoline drug action and resistance. The enhanced understanding of the biosynthetic pathway leading to haemozoin formation using functional proteomic tools and the mechanisms through which existing antimalarial drugs affect Plasmodium haeme chemistry will help design improved chaemotherapeutic agents.     

Brain metabolic markers reflect susceptibility status in cytokine gene knockout mice with murine cerebral malaria

Treatment of cerebral malaria, a complication of the world's most significant parasitic disease, remains problematic due to lack of understanding of its pathogenesis. Metabolic changes, along with cytokine expression alterations and blood cell sequestration in the brain, have previously been reported during severe disease in human infection and mouse models leading to the "cytopathic hypoxia" and "sequestration" theories of pathogenesis. Here, to determine the robustness of the metabolic changes and their relationship to disease development, we investigated changes in cerebral metabolic markers in a mouse model of cerebral malaria (CM) in wildtype (C57BL/6) and cytokine knockout (TNF(-/-), IFNgamma(-/-) and LTalpha(-/-)) mice using multinuclear magnetic resonance spectroscopy. Mice susceptible to CM (wildtype, TNF(-/-)) showed decreased cerebral glucose use, decreased Krebs cycle metabolism and decreased high-energy phosphates. Conversely, mice resistant to CM (IFNgamma(-/-), LTalpha(-/-)) showed little sign of these effects, despite identical levels of parasitemia. Previously reported changes in lactate were shown to be strain dependent. Elevated glutamine and decreased phosphorylation potential emerged as robust metabolic markers of susceptibility, further implicating the trytophan/NAD(+) pathway in disease development. Thus these metabolic changes are firmly linked both to the immune system response to malaria and to the occurrence of pathogenic changes in experimental CM.     

Inhibition of Plasmodium sporozoites infection by targeting the host cell

There is a great need of new drugs against malaria because of the increasing spread of parasite resistance against the most commonly used drugs in the field. We found that monensin, a common veterinary antibiotic, has a strong inhibitory effect in Plasmodium berghei and Plasmodium yoelii sporozoites hepatocyte infection in vitro. Infection of host cells by another apicomplexan parasite with a similar mechanism of host cell invasion, Toxoplasma tachyzoites, was also inhibited. Treatment of mice with monensin abrogates liver infection with P. berghei sporozoites in vivo. We also found that at low concentrations monensin inhibits the infection of Plasmodium sporozoites by rendering host cells resistant to infection, rather than having a direct effect on sporozoites. Monensin effect is targeted to the initial stages of parasite invasion of the host cell with little or no effect on development, suggesting that this antibiotic affects an essential host cell component that is required for Plasmodium sporozoite invasion.     

High gametocyte complexity and mosquito infectivity of Plasmodium falciparum in the Gambia

The purpose of this work was to determine the infectivity to mosquitoes of genetically diverse Plasmodium falciparum clones seen in natural infections in the Gambia. Two principal questions were addressed: (i) how infectious are gametocytes of sub-patent infections, particularly at the end of the dry season; and (ii) are all clones in multiclonal infections equally capable of infecting mosquitoes? The work was carried out with two cohorts of infected individuals. Firstly, a group of 31 P. falciparum-infected people were recruited in the middle of the dry season (May, 2003), then examined for P. falciparum at the beginning (August 2003) and middle (October, 2003) of the transmission season. On each occasion, we examined the genotypes of asexual forms and gametocytes by PCR and RT-PCR, as well as their infectivity to Anopheles gambiae using membrane feeds. One individual gave rise to infected mosquitoes in May, and two in August. Different gametocyte genotypes co-existed in the same infection and fluctuated over time. The mean multiplicity of infection was 1.4, 1.7 and 1.5 clones in May, August and October, respectively. Second, a group of patients undergoing drug-treatment during August 2003 was tested for asexual and gametocyte genotypes and their infectivity to mosquitoes. Forty-three out of 100 feeds produced infections. The genetic complexity of the parasites in mosquitoes was sometimes greater than that detectable in the blood on which the mosquitoes had fed. This suggested that gametocytes of clones existing in the blood below PCR detection limits at the time of the feed were at least as infectious to the mosquitoes as the more abundant clones. These findings emphasise the crucial role of gametocyte complexity and infectivity in generating the remarkable diversity of P. falciparum genotypes seen in infected people, even in an area of seasonal transmission.     

TNF family members and malaria: Old observations, new insights and future directions

Tumor necrosis factor (TNF) has long been recognized to promote malaria parasite killing, but also to contribute to the development of severe malaria disease. The precise molecular mechanisms that influence these different outcomes in malaria patients are not well understood, but the virulence and drug-resistance phenotype of malaria parasites and the genetic background and age of patients are likely to be important determinants. In the past few years, important roles for other TNF family members in host immune responses to malaria parasites and the induction of disease pathology have been discovered. In this review, we will summarize these more recent findings and highlight major gaps in our current knowledge. We will also discuss future research strategies that may allow us to better understand the sometimes subtle and intricate effects of TNF family molecules during malaria infection.     

Plasmodium berghei XAT: contribution of gammadelta T cells to host defense against infection with blood-stage nonlethal malaria parasite

We examined a potential role of gammadelta T cells in protective immunity to blood-stage Plasmodium berghei XAT infection. Plasmodium berghei XAT is an attenuated variant of the lethal strain P. berghei NK65 and its infection is self-resolving in immune competent mice. To determine whether gammadelta T cells are essential for the resolution of P. berghei XAT malaria, mice were depleted of gammadelta T cells with anti-TCRgammadelta antibody treatment. Although mice that had received control antibody resolved infections, mice received anti-TCRgammadelta antibody could not control their infections and eventually died. Spleen cells from infected mice produced IFN-gamma and nitric oxide (NO) within the first week of infection, however, levels of IFN-gamma and NO in gammadelta T cell-depleted mice were significantly lower than in control mice. To examine whether gammadelta T cells are involved in the antibody production, malarial-specific antibodies of the various isotypes were measured in the sera of gammadelta T cell-depleted mice and control mice. Serum levels of IgG2a, which was known to be a protective antibody in P. berghei XAT malaria, were significantly lower in gammadelta T cell-depleted mice than in control mice, whereas levels of IgG1 were comparable to those in control mice. Our results indicated that the presence of the gammadelta T cell subset was essential for resolution of blood-stage P. berghei XAT malaria and played a modulatory role in the development of Th1 response and host defense against this malarial parasites.     

Plasmodium berghei: antiparasitic effects of orally administered hypoestoxide in mice

Hypoestoxide (HE) is a diterpene isolated from Hypoestes rosea (Acanthaceae), a plant indigenous to Nigeria. Previous studies demonstrated that HE exhibited potent anti-inflammatory and anti-cancer activities in well established animal models but weak in vitro activities in both the anti-inflammation and anti-cancer in vitro screening systems. We now report a similar observation in the in vitro and in vivo screening systems for antimalarial activity. The results indicate that while HE exhibits a relatively weak in vitro activity (IC(50) = 10 microM versus 0.11 microM for chloroquine) against different strains of cultured P. falciparum parasites, the dose of HE required to reduce parasitemia by 90% in Plasmodium berghei-infected mice, is much lower than standard antimalaria drugs (SD(90) = 250 microg/kg versus 5mg/kg for chloroquine). Furthermore, lower doses of HE were much more effective than higher doses in inhibiting parasite development. The implications of these findings are discussed.     

Morphology and kinetics of the three distinct phases of red blood cell invasion by Plasmodium falciparum merozoites

The invasion of red blood cells (RBCs) is an essential event in the life cycle of all malaria-causing Plasmodium parasites; however, there are major gaps in our knowledge of this process. Here, we use video microscopy to address the kinetics of RBC invasion in the human malaria parasite Plasmodium falciparum. Under in vitro conditions merozoites generally recognise new target RBCs within 1 min of their release from their host RBC. Parasite entry ensues and is complete on average 27.6 s after primary contact. This period can be divided into two distinct phases. The first is an ∼11 s ‘pre-invasion’ phase that involves an often dramatic RBC deformation and recovery process. The second is the classical ‘invasion’ phase where the merozoite becomes internalised within the RBC in a ∼17 s period. After invasion, a third ‘echinocytosis’ phase commences when about 36 s after every successful invasion a dramatic dehydration-type morphology was adopted by the infected RBC. During this phase, the echinocytotic effect reached a peak over the next 23.4 s, after which the infected RBC recovered over a 5-11 min period. By then the merozoite had assumed an amoeboid-like state and was apparently free in the cytoplasm. A comparison of our data with that of an earlier study of the distantly related primate parasite Plasmodium knowlesi indicated remarkable similarities, suggesting that the kinetics of invasion are conserved across the Plasmodium genus. This study provides a morphological and kinetic framework onto which the invasion-associated physiological and molecular events can be overlaid.     

Plasmodium berghei XAT: protective 155/160 kDa antigens are located in parasitophorous vacuoles of schizont-stage parasite

Effective blood-stage malaria vaccine candidates have been mainly developed from the proteins in exposed locations on the parasite such as the surface of free merozoites or infected red blood cells. In the present study, we identified and localized novel protective antigens derived from the blood-stage of Plasmodium berghei XAT after establishment of hybridomas producing protective monoclonal antibodies (mAbs) against the parasites. The protective antigens were expressed in schizonts but not in trophozoites, and located in the parasitophorous vacuoles in the infected erythrocyte cytoplasm. The antigens, with molecular weight of 155/160 kDa, were not identical to any merozoite/schizont antigens that have been reported as target molecules recognized by mAbs developed to rodent malaria parasites. The characterization of new malarial antigenic targets of potentially protective antibody responses following infection would give us new insights for the selection of candidate antigens for malaria vaccine.