Beyond the microcirculation: sequestration of infected red blood cells and reduced flow in large draining veins in experimental cerebral malaria.

Sequestration of infected red blood cells (iRBCs) in the microcirculation is a hallmark of cerebral malaria (CM) in post-mortem human brains. It remains controversial how this might be linked to the different disease manifestations, in particular brain swelling leading to brain herniation and death. The main hypotheses focus on iRBC-triggered inflammation and mechanical obstruction of blood flow. Here, we test these hypotheses using murine models of experimental CM (ECM), SPECT-imaging of radiolabeled iRBCs and cerebral perfusion, MR-angiography, q-PCR, and immunohistochemistry. We show that iRBC accumulation and reduced flow precede inflammation. Unexpectedly, we find that iRBCs accumulate not only in the microcirculation but also in large draining veins and sinuses, particularly at the rostral confluence. We identify two parallel venous streams from the superior sagittal sinus that open into the rostral rhinal veins and are partially connected to infected skull bone marrow. The flow in these vessels is reduced early, and the spatial patterns of pathology correspond to venous drainage territories. Our data suggest that venous efflux reductions downstream of the microcirculation are causally linked to ECM pathology, and that the different spatiotemporal patterns of edema development in mice and humans could be related to anatomical differences in venous anatomy.

Survey of Hepatocystis parasites of fruit bats in the Amurum forest reserve, Nigeria, identifies first host record for Rousettus aegyptiacus.

Parasites of the genus Hepatocystis are close relatives of Plasmodium that frequently infect epauletted fruit bats across West and East Africa. Our understanding of susceptible hosts and prevalence of infection of Hepatocystis remains fragmented. Non-invasive sampling of bat assemblages in representative habitats critically contribute to haemosporidian parasite distribution maps. Here, we report on a survey of Hepatocystis parasite infections in bats undertaken over two consecutive years in a protected area in Nigeria, where prevalence and diversity of bat-infecting haemosporidian parasites have not been studied. Microscopic examination of blood films in combination with PCR detection and sequencing revealed Hepatocystis infections with prevalences of 25% and 42% in the closely related epauletted fruit bats Epomophorus sp. and Micropteropus pusillus. For the first time, mature Hepatocystis gametocytes were identified in one Egyptian fruit bat (Rousettus aegyptiacus). This novel host record was confirmed by parasite and host genotyping and suggests that Hepatocystis parasites have a broader host distribution in African fruit bats than currently known.

Biochemistry of malaria parasite infected red blood cells by X-ray microscopy.

Red blood cells infected by the malaria parasite Plasmodium falciparum are correlatively imaged by tomography using soft X-rays as well as by scanning hard nano-X-ray beam to obtain fluorescence maps of various elements such as S and Fe. In this way one can deduce the amount of Fe bound either in hemoglobin or in hemozoin crystals in the digestive vacuole of the malaria parasite as well as determine the hemoglobin concentrations in the cytosols of the red blood cell and of the parasite. Fluorescence map of K shows that in the parasite's schizont stage the K concentration in the red blood cell cytosol is diminished by a factor of seven relative to a pristine red blood cell but the total amount of K in the infected red blood cell is the same as in the pristine red blood cell.

Evidence of cross-stage CD8+ T cell epitopes in malaria pre-erythrocytic and blood stage infections.

Malaria parasites have a complex, multistage life cycle and there is a widely held view that each stage displays a distinct set of antigens presented to the immune system. Yet, molecular analysis of malaria parasites suggests that many putative antigenic targets are shared amongst the different stages. The specificities of these cross-stage antigens and the functions of the immune responses they elicit are poorly characterized. It is well-known that CD8+ T cells play opposing immune functions following Plasmodium berghei (Pb) infection of C57BL/6 mice. Whilst these cells play a crucial role in protective immunity against pre-erythrocytic stages, they are implicated in the development of severe disease during blood stages. Recently, CD8+ T cell epitopes derived from proteins supposedly specific for either pre-erythrocytic or blood stages have been described. In this brief report, we have compiled and confirmed data that the majority of the mRNAs and/or proteins from which these epitopes are derived display expression across pre-erythrocytic and blood stages. Importantly, we provide evidence of cross-stage immune recognition of the majority of these CD8+ T cell epitopes. Hence, our findings provide a resource to further examine the relevance of antigen-specific cross-stage responses during malaria infections.

Batf3 deficiency proves the pivotal role of CD8α+ dendritic cells in protection induced by vaccination with attenuated Plasmodium sporozoites.

Increasing evidence indicates that hepatic CD8α+ dendritic cells (DCs) are important antigen cross-presenting cells (APC) involved in the priming of protective CD8+ T-cell responses induced by live-attenuated Plasmodium sporozoites. Experimental proof for a critical role of CD8α+ DCs in protective pre-erythrocytic malaria immunizations has pivotal implications for vaccine development, including improved vectored subunit vaccines. Employing Batf3-/- mice, which lack functional CD8α+ DCs, we demonstrate that deficiency of these particular APCs completely abolishes protection and corresponding signatures of vaccine-induced immunity. We show that in wild-type, but not in Batf3-/- , mice CD8α+ DCs accumulate in the liver after immunization with live irradiation-attenuated P. berghei sporozoites. IFN-γ production by Plasmodium antigen-specific CD8+ T cells is dependent on functional Batf3. In addition, our results demonstrate that the dysfunctional cDC-CD8+ T-cell axis correlates with MHC class II upregulation on splenic CD8α- DCs. Collectively, these findings underscore the essential role of CD8α+ DCs in robust protection induced by experimental live-attenuated malaria vaccines.

Signatures of malaria vaccine efficacy in ageing murine immune memory.

Malaria transmission occurs by mosquito bite. Thereafter, Plasmodium sporozoites specifically invade the liver, where they develop into thousands of merozoites that initiate blood-stage infection and clinical malaria. The pre-erythrocytic phase of a Plasmodium infection is the target of experimental whole-parasite vaccines against malaria. Repeated immunizations with high doses of live, metabolically active sporozoites can induce protracted protection against Plasmodium reinfection. Parasites lacking a Plasmodium-specific apicoplast protein, termed PALM, arrest very late during intrahepatic development just prior to liver merozoite release and can elicit sterile protection with two immunization doses only. In this report, we show in the robust Plasmodium berghei-C57BL/6 model that partial protection extends beyond 1 year after the last immunization. In ageing mice, intracellular cytokine staining of Plasmodium peptide-stimulated intrahepatic CD8+ T cells revealed elevated levels of interferon gamma in vaccinated mice. We conclude that antigen-specific T cells persist in the target organ and are critical signatures of lasting protection. Our data also support the notions that memory T-cell responses generated early in life remain largely intact well into old age and that murine Plasmodium vaccination and infection models are suitable to study the mechanisms of maintenance and efficiency of adaptive immunity during immunosenescence.

Hitting malaria before it hurts: attenuated Plasmodium liver stages.

Continuous natural exposure to Plasmodium transmission by infectious Anopheles mosquitoes leads to a gradual acquisition of immunological competence against malaria. The partial immunity, observed in adolescents and adults living in endemic areas, reduces morbidity and mortality without preventing parasite infection. In experimental animal models, long-lasting sterilizing immunity can be achieved with genetically attenuated Plasmodium liver stages. Can these findings be translated to accomplish sterile protection against natural malaria transmission in the high-risk group, young infants in sub-Saharan Africa?

Exploring the transcriptome of the malaria sporozoite stage.

Most studies of gene expression in Plasmodium have been concerned with asexual and/or sexual erythrocytic stages. Identification and cloning of genes expressed in the preerythrocytic stages lag far behind. We have constructed a high quality cDNA library of the Plasmodium sporozoite stage by using the rodent malaria parasite P. yoelii, an important model for malaria vaccine development. The technical obstacles associated with limited amounts of RNA material were overcome by PCR-amplifying the transcriptome before cloning. Contamination with mosquito RNA was negligible. Generation of 1,972 expressed sequence tags (EST) resulted in a total of 1,547 unique sequences, allowing insight into sporozoite gene expression. The circumsporozoite protein (CS) and the sporozoite surface protein 2 (SSP2) are well represented in the data set. A BLASTX search with all tags of the nonredundant protein database gave only 161 unique significant matches (P(N) < or = 10(-4)), whereas 1,386 of the unique sequences represented novel sporozoite-expressed genes. We identified ESTs for three proteins that may be involved in host cell invasion and documented their expression in sporozoites. These data should facilitate our understanding of the preerythrocytic Plasmodium life cycle stages and the development of preerythrocytic vaccines.

Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing.

Processing of integral membrane proteins in order to liberate active proteins is of exquisite cellular importance. Examples are the processing events that govern sterol regulation, Notch signaling, the unfolded protein response, and APP fragmentation linked to Alzheimer's disease. In these cases, the proteins are thought to be processed by regulated intramembrane proteolysis, involving site-specific, membrane-localized proteases. Here we show that two homologous yeast transcription factors SPT23 and MGA2 are made as dormant ER/nuclear membrane-localized precursors and become activated by a completely different mechanism that involves ubiquitin/proteasome-dependent processing. SPT23 and MGA2 are relatives of mammalian NF-kappaB and control unsaturated fatty acid levels. Intriguingly, proteasome-dependent processing of SPT23 is regulated by fatty acid pools, suggesting that the precursor itself or interacting partners are sensors of membrane composition or fluidity.

A novel protein modification pathway related to the ubiquitin system.

Ubiquitin conjugation is known to target protein substrates primarily to degradation by the proteasome or via the endocytic route. Here we describe a novel protein modification pathway in yeast which mediates the conjugation of RUB1, a ubiquitin-like protein displaying 53% amino acid identity to ubiquitin. We show that RUB1 conjugation requires at least three proteins in vivo. ULA1 and UBA3 are related to the N- and C-terminal domains of the E1 ubiquitin-activating enzyme, respectively, and together fulfil E1-like functions for RUB1 activation. RUB1 conjugation also requires UBC12, a protein related to E2 ubiquitin-conjugating enzymes, which functions analogously to E2 enzymes in RUB1-protein conjugate formation. Conjugation of RUB1 is not essential for normal cell growth and appears to be selective for a small set of substrates. Remarkably, CDC53/cullin, a common subunit of the multifunctional SCF ubiquitin ligase, was found to be a major substrate for RUB1 conjugation. This suggests that the RUB1 conjugation pathway is functionally affiliated to the ubiquitin-proteasome system and may play a regulatory role.

The ubiquitin-like proteins SMT3 and SUMO-1 are conjugated by the UBC9 E2 enzyme.

The ubiquitin-like protein SMT3 from Saccharomyces cerevisiae and SUMO-1, its mammalian homolog, can be covalently attached to other proteins posttranslationally. Conjugation of ubiquitin requires the activities of ubiquitin-activating (E1) and -conjugating (E2) enzymes and proceeds via thioester-linked enzyme-ubiquitin intermediates. Herein we show that UBC9, one of the 13 different E2 enzymes from yeast, is required for SMT3 conjugation in vivo. Moreover, recombinant yeast and mammalian UBC9 enzymes were found to form thioester complexes with SMT3 and SUMO-1, respectively. This suggests that UBC9 functions as an E2 in a SMT3/SUMO-1 conjugation pathway analogous to ubiquitin-conjugating enzymes. The role of yeast UBC9 in cell cycle progression may thus be mediated through its SMT3 conjugation activity.

Identification of a novel family of ubiquitin-conjugating enzymes with distinct amino-terminal extensions.

The ubiquitin/proteasome system is the main eukaryotic nonlysosomal protein degradation system. Substrate selectivity of this pathway is thought to be mediated in part by members of a large family of ubiquitin-conjugating (E2) enzymes, which catalyze the covalent attachment of ubiquitin to proteolytic substrates. E2 enzymes have a conserved approximately 150-residue so-called UBC domain, which harbors the cysteine residue required for enzyme-ubiquitin thioester formation. Some E2 enzymes possess additional carboxyl-terminal extensions that are involved in substrate specificity and intracellular localization of the enzyme. Here we describe a novel family of E2 enzymes from higher eukaryotes (Drosophila, mouse, and man) that have amino-terminal extensions but lack carboxyl-terminal extensions. We have identified four different variants of these enzymes that have virtually identical UBC domains (94% identity) but differ in their amino-terminal extensions. In yeast, these enzymes can partially complement mutants deficient in the UBC4 E2 enzyme. This indicates that members of this novel E2 family may operate in UBC4-related proteolytic pathways.