The plastid (apicoplast) of the malaria-causing parasite Plasmodium falciparum was derived via a secondary endosymbiotic process. As in other secondary endosymbionts, numerous genes for apicoplast proteins are located in the nucleus, and the encoded proteins are targeted to the organelle courtesy of a bipartite N-terminal extension. The first part of this leader sequence is a signal peptide that targets proteins to the secretory pathway. The second, so-called transit peptide region is required to direct proteins from the secretory pathway across the multiple membranes surrounding the apicoplast. In this paper we perform a pulse-chase experiment and N-terminal sequencing to show that the transit peptide of an apicoplast-targeted protein is cleaved, presumably upon import of the protein into the apicoplast. We identify a gene whose product likely performs this cleavage reaction, namely a stromal-processing peptidase (SPP) homologue. In plants SPP cleaves the transit peptides of plastid-targeted proteins. The P. falciparum SPP homologue contains a bipartite N-terminal apicoplast-targeting leader. Interestingly, it shares this leader sequence with a ⌬-aminolevulinic acid dehydratase homologue via an alternative splicing event.Plasmodium spp., the causative agents of malaria, belong to a family of intracellular parasites called the Apicomplexa. Plasmodium infects approximately 300 million people annually, causing over 1 million deaths, the great majority of which are caused by one species, Plasmodium falciparum (1). P. falciparum infects both humans and mosquitoes during its life cycle, with the pathogenic part of this cycle occurring predominantly in the erythrocytes of humans. The discovery of a non-photosynthetic plastid (the apicoplast) in the Apicomplexa has opened up a new area of anti-malarial drug targets (2, 3). However, the rational development of drugs targeting the apicoplast requires knowledge of apicoplast function. Preliminary studies indicate that the apicoplast is a site of fatty acid and isoprenoid biosynthesis (4 -6), and drugs targeting these pathways have been shown to kill P. falciparum (4, 6, 7).Like plant plastids, the apicoplast contains a reduced bacterial-like genome (8), from which a small number of proteins are expressed. The great majority of apicoplast proteins, as in plant plastids, are encoded in the nucleus and must be post-translationally targeted to the plastid. In plants, nuclear-encoded plastid proteins require a cleavable, N-terminal sequence called the transit peptide, which directs these proteins across the two membranes surrounding plant plastids (for reviews, see Refs. 9 and 10). Once in the plastid stroma, this transit peptide is cleaved by a stromal-processing peptidase (SPP 1 ; Refs. 11 and 12). Apicoplasts, however, are bound by four membranes (Ref. 2, but see Ref. 13), and proteins targeted to this organelle have been shown to require a bipartite N-terminal leader sequence (5,14,15). By fusing these N-terminal leader sequences to green fluorescent reporter protein (GFP)...

High, early IFN-gamma production by live parasite-stimulated peripheral blood mononuclear cells is a correlate of immunity to symptomatic malaria in Papua New Guinean children, and natural killer-like gammadelta T cells may contribute to protection.

No abstract

Rapid production of interferon‐γ (IFN‐γ) in response to malaria by the innate immune system may determine resistance to infection, or inflammatory disease. However, conflicting reports exist regarding the identity of IFN‐γ‐producing cells that rapidly respond to Plasmodium falciparum. To clarify this area, we undertook detailed phenotyping of IFN‐γ‐producing cells across a panel of naive human donors following 24‐h exposure to live schizont‐infected red blood cells (iRBC). Here, we show that NK cells comprise only a small proportion of IFN‐γ‐responding cells and that IFN‐γ production is unaffected by NK cell depletion. Instead, γδ‐T cells represent the predominant source of innate IFN‐γ, with the majority of responding γδ‐T cells expressing NK receptors. Malaria‐responsive γδ‐T cells more frequently expressed NKG2A compared to non‐responding γδ‐T cells, while non‐responding γδ‐T cells more frequently expressed CD158a/KIR2DL1. Unlike long‐term γδ‐T cell responses to iRBC, αβ‐T cell help was not required for innate γδ‐T cell responses. Diversity was observed among donors in total IFN‐γ output. This was positively associated with CD94 expression on IFN‐γ+ NK‐like γδ‐T cells. Applied to longitudinal cohort studies in endemic regions, similar comparative phenotyping should allow assessment of the contribution of diverse cell populations and regulatory receptors to risk of infection and disease.

SummaryHematopoiesis is a multistage process involving the differentiation of stem and progenitor cells into distinct mature cell lineages. Here we present Haemopedia, an atlas of murine gene-expression data containing 54 hematopoietic cell types, covering all the mature lineages in hematopoiesis. We include rare cell populations such as eosinophils, mast cells, basophils, and megakaryocytes, and a broad collection of progenitor and stem cells. We show that lineage branching and maturation during hematopoiesis can be reconstructed using the expression patterns of small sets of genes. We also have identified genes with enriched expression in each of the mature blood cell lineages, many of which show conserved lineage-enriched expression in human hematopoiesis. We have created an online web portal called Haemosphere to make analyses of Haemopedia and other blood cell transcriptional datasets easier. This resource provides simple tools to interrogate gene-expression-based relationships between hematopoietic cell types and genes of interest.

The role of early to intermediate Plasmodium falciparum-induced cellular responses in the development of clinical immunity to malaria is not well understood, and such responses have been proposed to contribute to both immunity and risk of clinical malaria episodes. To investigate whether P. falciparum-induced cellular responses are able to function as predictive correlates of parasitological and clinical outcomes, we conducted a prospective cohort study of children (5 to 14 years of age) residing in a region of Papua New Guinea where malaria is endemic Live, intact P. falciparum-infected red blood cells were applied to isolated peripheral blood mononuclear cells obtained at baseline. Cellular cytokine production, including production of interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor (TNF) (formerly tumor necrosis factor alpha), and gamma interferon (IFN-␥), was measured, and the cellular source of key cytokines was investigated. Multicytokine models revealed that increasing P. falciparum-induced IL-6 production was associated with an increased incidence of P. falciparum clinical episodes (incidence rate ratio [ Individuals living in regions of moderate to high malaria endemicity slowly acquire clinical immunity to Plasmodium falciparum throughout their lives in an age-and exposuredependent manner (3,29). This immunity enables them to control parasite replication at densities below that which induces clinical symptoms (29,46). Both antibody-dependent and T-cell-dependent acquired immune responses have been shown to play an important role in the development of clinical immunity (13,29). The role of early to intermediate cellular responses, however, is less well understood, and such responses have been proposed to contribute to both immunity and risk of clinical malaria episodes (45,49,51).Early cellular immune responses are rapidly initiated during malaria infection and are thought to play an important role both in limiting initial parasite replication and in directly shaping subsequent adaptive immune responses (45,49,51). However, the overproduction or inappropriate regulation of both proinflammatory cytokines, such as interleukin-1 (IL-1), IL-6, gamma interferon (IFN-␥), and tumor necrosis factor (TNF) (formerly tumor necrosis factor alpha), and anti-inflammatory cytokines, such as IL-10, IL-4, and transforming growth factor ␤ (TGF-␤), may also lead to localized and systemic inflammation and has been associated with symptomatic and severe malaria (7,45). The clinical outcome of an infection may thus depend on the appropriate induction and counterregulation of both pro-and anti-inflammatory cytokine secretion. Understanding how this network of P. falciparum-induced cellular responses is associated with immunity and risk of clinical disease in malaria exposed-children could provide important insights for the development of effective vaccines.Studies investigating the association between P. falciparumor antigen-induced secretion of cytokines from peripheral blood mononuclear cells (PBMCs) and prosp...

Caspases are best known for their role in apoptosis. More recently, they have gained prominence as critical mediators of innate immune responses. The so-called ‘inflammatory caspases’ include human caspase-1, -4, -5 and -12 and murine caspase-1, -11 and -12. Of these, caspase-1 is best characterized and serves as the prototype for our understanding of the processing, activation and function of inflammatory caspases. Like their apoptotic counterparts, inflammatory caspases are produced as inactive zymogens and require activation to become proteolytically active. Caspase-1 is activated within the inflammasome, a large cytosolic protein complex that is induced by a growing number of endogenous, microbial, chemical or environmental stimuli. The importance of caspase-1 in initiating innate immune responses is demonstrated by its role in cleaving pro-IL-1β and pro-IL-18 to their biologically active forms. New functions have also been implicated, as these proteases and the mechanisms underlying their activation and regulation emerge as important mediators of human health and disease.

Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is a variable antigen expressed by P. falciparum, the malarial parasite. PfEMP-1, present on the surface of infected host erythrocytes, mediates erythrocyte binding to vascular endothelium, enabling the parasite to avoid splenic clearance. In addition, PfEMP-1 is proposed to regulate host immune responses via interactions with the CD36 receptor on antigen-presenting cells. We investigated the immunoregulatory function of PfEMP-1 by comparing host cell responses to erythrocytes infected with either wild-type parasites or transgenic parasites lacking PfEMP-1. We showed that PfEMP-1 suppresses the production of the cytokine interferon-gamma by human peripheral blood mononuclear cells early after exposure to P. falciparum. Suppression of this rapid proinflammatory response was CD36 independent and specific to interferon-gamma production by gammadelta-T, NK, and alphabeta-T cells. These data demonstrate a parasite strategy for downregulating the proinflammatory interferon-gamma response and further establish transgenic parasites lacking PfEMP-1 as powerful tools for elucidating PfEMP-1 functions.