We report the cloning of a Leishmania chagasi antigen gene and an evaluation of leishmaniasis patient antibody responses to the recombinant protein, rK39. rK39 contains a 39-amino acid repeat that is part of a 230-kDa protein predominant in L. chagasi tissue amastigotes. Sequence analyses showed this protein, LcKin, to be related to the kinesin superfamily of motor proteins. Southern blot analyses demonstrated LcKin-related sequences in seven species of Leishmania, with conservation of the repeat between L. chagasi and Leishmania donovani. Serological evaluation revealed that 98% (56 of 57) of Brazilian and 100% (52 of 52) of Sudanese visceral leishmaniasis patients have high antibody levels to the rK39 repeat. Detectable anti-K39 antibody was virtually absent in cutaneous and mucosal leishmaniasis patients and in individuals infected with Trypanosoma cruzi. The data show that rK39 may replace crude parasite antigens as a basis for serological diagnosis of visceral leishmaniasis.

Summary Intracellular pathogens have devised mechanisms to exploit their host cells to ensure their survival and replication. The malaria parasite Plasmodium falciparum relies on an exchange of metabolites with the host for proliferation. We describe the first mass spectrometry-based metabolomic analysis of the parasite throughout its 48-hour intraerythrocytic developmental cycle. Our results reveal a general modulation of metabolite levels by the parasite, with numerous metabolites varying in phase with the developmental cycle. Others differed from uninfected cells irrespective of the developmental stage. Among these was extracellular arginine, which was specifically converted to ornithine by the parasite. To identify the biochemical basis for this effect, we disrupted the plasmodium arginase gene in the rodent malaria model P. berghei. These parasites were viable but did not convert arginine to ornithine. Our results suggest that systemic arginine depletion by the parasite may be a factor in human malarial hypoargininemia associated with cerebral malaria pathogenesis.

The diagnosis of visceral leishmaniasis (VL), a serious and often fatal parasitic disease caused by members of the Leishmania donovani complex, remains problematic. Current methods rely on clinical criteria, parasite identification in aspirate material, and serology. The latter methods use crude antigen preparations lacking in specificity. A previously described cloned antigen, rK39, of Leishmania specific for all members of the L. donovani complex (L. chagasi, L. donovani, L. infantum) was very useful in the serodiagnosis by ELISA of both human and canine VL. The present study demonstrated that rK39 seroreactivity correlated with active disease. The sera from early or self-healing infected subjects reacted with leishmanial lysate and were generally nonreactive with rK39. These data demonstrate the utility of rK39 in the serodiagnosis of VL and as an indicator of active disease.

Among the several new antimalarials discovered over the past decade are at least three clinical candidate drugs, each with a distinct chemical structure, that disrupt Na+ homeostasis resulting in a rapid increase in intracellular Na+ concentration ([Na+]i) within the erythrocytic stages of Plasmodium falciparum. At present, events triggered by Na+ influx that result in parasite demise are not well-understood. Here we report effects of two such drugs, a pyrazoleamide and a spiroindolone, on intraerythrocytic P. falciparum. Within minutes following the exposure to these drugs, the trophozoite stage parasite, which normally contains little cholesterol, was made permeant by cholesterol-dependent detergents, suggesting it acquired a substantial amount of the lipid. Consistently, the merozoite surface protein 1 and 2 (MSP1 and MSP2), glycosylphosphotidylinositol (GPI)-anchored proteins normally uniformly distributed in the parasite plasma membrane, coalesced into clusters. These alterations were not observed following drug treatment of P. falciparum parasites adapted to grow in a low [Na+] growth medium. Both cholesterol acquisition and MSP1 coalescence were reversible upon the removal of the drugs, implicating an active process of cholesterol exclusion from trophozoites that we hypothesize is inhibited by high [Na+]i. Electron microscopy of drug-treated trophozoites revealed substantial morphological changes normally seen at the later schizont stage including the appearance of partial inner membrane complexes, dense organelles that resemble “rhoptries” and apparent nuclear division. Together these results suggest that [Na+]i disruptor drugs by altering levels of cholesterol in the parasite, dysregulate trophozoite to schizont development and cause parasite demise.

Morbidity and mortality due to Plasmodium falciparum and Plasmodium vivax, the two predominant human malaria parasites, result during the asexual development and replication of these protozoan parasites within erythrocytes (RBCs) (44). To reduce this burden on nearly half of the world's population, several malaria vaccine strategies are being pursued (28,40,53). Blood-stage vaccines are being developed to reduce parasite load and/or prevent life-threatening complications of malaria once parasites are replicating within RBCs. The single most feasible strategy for blood-stage malaria is to immunize with subunit vaccines that induce high titers of antibodies that neutralize extracellular merozoites and prevent the invasion of erythrocytes (2,25,31,40). The multiple receptor-ligand interactions and alternate redundant pathways involved in the merozoite invasion of RBCs combined with the polymorphism of vaccine candidate antigens present a challenge for vaccine design (2,25,26).P. falciparum merozoite surface protein 1 (MSP-1)

Knowledge of the dynamic features of the processes driven by malaria parasites in the spleen is lacking. To gain insight into the function and structure of the spleen in malaria, we have implemented intravital microscopy and magnetic resonance imaging of the mouse spleen in experimental infections with non-lethal (17X) and lethal (17XL) Plasmodium yoelii strains. Noticeably, there was higher parasite accumulation, reduced motility, loss of directionality, increased residence time and altered magnetic resonance only in the spleens of mice infected with 17X. Moreover, these differences were associated with the formation of a strain-specific induced spleen tissue barrier of fibroblastic origin, with red pulp macrophage-clearance evasion and with adherence of infected red blood cells to this barrier. Our data suggest that in this reticulocyte-prone non-lethal rodent malaria model, passage through the spleen is different from what is known in other Plasmodium species and open new avenues for functional/structural studies of this lymphoid organ in malaria.

A gene sequence encoding an immunodominant protein with a repetitive epitope from the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, was cloned and expressed. The identified 10-amino acid repeat is present within a high-molecular-weight trypomastigote antigen that appears specific to and conserved among T. cruzi isolates. More importantly, greater than 95% of T. cruzi infection sera, including both chronic and acute Chagas disease, contained elevated levels of antibody to a 15-amino acid synthetic peptide bearing the repetitive B-cell epitope. Considering the wide diversity of T. cruzi parasites, as well as the broad spectrum of clinical manifestations of Chagas disease, such a prevalent immune response among patients is significant and applicable to the control of Chagas disease through the diagnosis of T. cruzi infection.

ABSTRACTThe C-terminal 19-kDa domain of merozoite surface protein 1 (MSP119) is the target of protective antibodies but alone is poorly immunogenic. Previously, using thePlasmodium yoeliimurine model, we fusedP. yoeliiMSP119(PyMSP119) with full-lengthP. yoeliimerozoite surface protein 8 (MSP8). Upon immunization, the MSP8-restricted T cell response provided help for the production of high and sustained levels of protectivePyMSP119- andPyMSP8-specific antibodies. Here, we assessed the vaccine potential of MSP8 of the human malaria parasite,Plasmodium falciparum. Distinct fromPyMSP8,P. falciparumMSP8 (PfMSP8) contains an N-terminal asparagine and aspartic acid (Asn/Asp)-rich domain whose function is unknown. Comparative analysis of recombinant full-lengthPfMSP8 and a truncated version devoid of the Asn/Asp-rich domain,PfMSP8(ΔAsn/Asp), showed that both proteins were immunogenic for T cells and B cells. All T cell epitopes utilized mapped within rPfMSP8(ΔAsn/Asp). The dominant B cell epitopes were conformational and common to both rPfMSP8 and rPfMSP8(ΔAsn/Asp). Analysis of nativePfMSP8 expression revealed thatPfMSP8 is present intracellularly in late schizonts and merozoites. Following invasion,PfMSP8 is found distributed on the surface of ring- and trophozoite-stage parasites. Consistent with a low and/or transient expression ofPfMSP8 on the surface of merozoites,PfMSP8-specific rabbit IgG did not inhibit thein vitrogrowth ofP. falciparumblood-stage parasites. These studies suggest that the further development ofPfMSP8 as a malaria vaccine component should focus on the use ofPfMSP8(ΔAsn/Asp) and its conserved, immunogenic T cell epitopes as a fusion partner for protective domains of poor immunogens, includingPfMSP119.