疟原虫入侵红细胞的相关分子及其机制

疟原虫入侵宿主红细胞具有高度的种特异性,这些特异性的分子基础是疟原虫蛋白质与宿主红细胞表面蛋白质的相互作用.寻找参与疟原虫入侵红细胞的相关分子及其入侵机制是疟疾研究领域的热点.针对疟原虫不同种和虫株的实验研究并结合生物信息学分析结果表明:裂殖子表面蛋白、网织红细胞结合蛋白家族、红细胞结合蛋白家族以及动力蛋白等是参与疟原虫入侵红细胞的重要蛋白.该文对这方面的研究进展作了系统的综述,并阐述这些蛋白质在疟原虫入侵过程不同阶段中的作用.     

经化学和酶处理的红细胞膜对恶性疟原虫裂殖子入侵...

By treating chemically and enzymatically erythrocytic membrane to change its structure and properties, the recognition of merozoite to erythrocyte and the effect of erythrocyte skeleton protein on the invasion of P. falciparum (Fc c-1/HN) were studied. It was found that the invasion of merozoite into erythrocyte digested with trypsin was greatly decreased; merozoite invasion into the erythrocyte treated with neuraminidase or wheat germ agglutinin (WGA) was partially inhibited; erythrocytes treated with cross-linker diamide and depolymerizer colchicine, N-ethyl maleinimide (NEM) of skeleton protein had evident resistance to invasion; the invasion of merozoite into erythrocyte treated with 2.0 mM of NEM was completely blocked.     

一氧化氮对宿主抗疟原虫感染用机制的研究进展

一氧化氮( nitric oxide,NO)作为重要的细胞间信使,其本身及衍生物在呼吸、消化、循环、免疫、神经等全身多系统的生理、病理及有关临床疾病中起着重要作用.NO在宿主抗疟原虫感染的免疫应答过程中发挥重要作用.在疟原虫感染宿主的肝内期和蚊阶段,NO的保护性免疫作用已经得到肯定.在抗红内期疟原虫感染的免疫应答中,由...     

一氧化氮对宿主抗疟原虫感染作用机制的研究进展

一氧化氮（nitricoxide,NO）作为重要的细胞间信使,其本身及衍生物在呼吸、消化、循环、免疫、神经等全身多系统的生理、病理及有关临床疾病中起着重要作用。NO在宿主抗疟原虫感染的免疫应答过程中发挥重要作用。在疟原虫感染宿主的肝内期和蚊阶段,NO的保护性免疫作用已经得到肯定。在抗红内期疟原虫感染的免疫应答中,由于感染宿主的免疫状态、感染虫株的差异性以及疟原虫的发育阶段和NO产生部位的不同等多种因素的影响,使得NO生物学作用的发挥存在一定差异。NO可能通过直接杀伤作用、DNA损伤作用以及诱导凋亡等途径发挥杀伤疟原虫效应。该文就NO的作用机制研究进展做一综述。     

鼠疟原虫裂殖子进入红细胞后的早期超微结构演化

为了探讨疟原虫红内早期超微结构的演化，用透射电镜观察了伯氏和约氏疟原虫。裂殖子入侵后补围于内陷红细胞膜所构成的纳虫泡中，起初停留于浅层红细胞浆内，造成该处表面隆起。虫体浑圆，顶端结构很快消失，球形体与核和线粒体分离并萎缩。分离了的线粒体略显舒展，内质网扩大。核变长弯形，核周隙有部分地扩大。双层内膜渐与外膜分离，断裂、卷曲、以至消失。最后变成单层质膜的滋养体。早期滋养体的组织结构仍较精细，以后密度逐渐减低。胞浆内有个别多角形的大内质网池，质膜下出现小食物泡。然后虫体变扁平，边缘卷起，包围红细胞浆，终至封口融合，形成一个食物泡；接着在其旁出现消化泡和色素粒。长成更大的中期滋养体。     

疟原虫在人体内发育过程的研究进展

疟疾是由疟原虫引起的虫媒传染病,在全球尤其是热带地区肆虐流行,严重威胁着人类的健康.疟原虫主要在人类宿主体内发育繁殖,具有复杂的生命周期,包括疟原虫入侵、迁移、逃避免疫攻击、肝细胞内分裂增殖、入侵红细胞并在红细胞内大量繁殖、周期性释放等系列过程,最终导致疟疾患者症状周期性发作.本文重点围绕疟原虫(即子孢子、裂殖子)人体红细胞外、红细胞内入侵及发育过程的研究进展进行综述,为疟疾疫苗研发和疟疾防控救治提供参考.     

恶性疟原虫裂殖子表面蛋白疟疾疫苗的研究进展

疟疾是一种危害严重的虫媒传染病，在疟原虫对抗疟药物的耐药性及媒介蚊对杀虫剂耐药性日益增强的今天，寻求一种新型，高效，安全的抗疟途径－抗疟疾疫苗的研制备受科研人员的关注。本对恶性疟原虫裂殖子表面蛋白抗疟疾疫苗的研究进展作一概述。     

Malaria parasite invasion: interactions with the red cell membrane

The capacity to invade red cells is central to the biology of malaria parasites; both asexual multiplication and reinfection of the definitive mosquito host depend upon intraerythrocytic stages. The invasion process is complex. The briefly free merozoite specifically recognizes and adheres to ligands on the red cell surface, then alters the red cell membrane to produce an invagination into which it moves, and so becomes enclosed in a membrane-bound parasitophorous vacuole. Here we assess new evidence that bears on our understanding of this process. This has come from sources including biochemical and ultrastructural studies of the specialized surface and organelles of merozoites, from in vitro invasion studies using naturally refractory or artificially modified red cells, and from structural, chemical, and immunological analyses of the newly parasitized cell.     

The interaction between the malaria parasite and the red cell membrane

The malaria parasite intimately interacts with the host red cell membrane throughout the cycle of invasion and intracellular development. Direct interaction between the merozoite surface and the red cell membrane involves specific binding between the surface components of both cells, which leads to the subsequent endocytotic process still incompletely understood. Intracellular development of the parasite is accompanied by various changes in the structure and function of red cell membrane components. Some changes may benefit parasite survival while others trigger host immune response. An understanding of both the direct interaction between the surface components of the parasite and the red cell during invasion, and the subsequent changes in the red cell membrane following invasion, should lead to better ways of controlling malaria.     

Polyethylene glycol-coated red blood cells fail to bind glycophorin A-specific antibodies and are impervious to invasion by the Plasmodium falciparum malaria parasite

This study was designed to assess the binding of glycophorin A-specific antibodies to polyethylene glycol (PEG)-modified red blood cells (RBCs) and evaluate their resistance to invasion by Plasmodium falciparum malaria parasites. RBCs were conjugated with a range of concentrations (0.05 to 7.5 mM) of activated PEG derivatives of either 3.35 or 18.5 kd molecular mass. The binding of glycophorin A-specific antibodies was assessed by hemagglutination and flow cytometry. PEG-modified RBCs were assessed for their ability to form rosettes around Chinese hamster ovary (CHO) cells transiently expressing the glycophorin A binding domain of EBA-175, a P falciparum ligand crucial to RBC invasion. PEG-RBCs were also tested for their ability to be invaded by the malaria parasite. RBCs coated with 3.35 and 18.5 kd PEG demonstrated a dose-dependent inhibition of glycophorin A-specific antibody binding, CHO cell rosetting, and P falciparum invasion. These results indicate that glycophorin A epitopes responsible for antibody and parasite binding are concealed by PEG coating, rendering these cells resistant to P falciparum invasion. These studies confirm the effectiveness of PEG modification for masking RBC-surface glycoproteins. This may provide a means to prevent alloimmunization in the setting of RBC transfusion and suggests a novel method to enhance the effectiveness of exchange transfusion for the treatment of cerebral malaria.     

Structure of the MTIP-MyoA complex, a key component of the malaria parasite invasion motor

The causative agents of malaria have developed a sophisticated machinery for entering multiple cell types in the human and insect hosts. In this machinery, a critical interaction occurs between the unusual myosin motor MyoA and the MyoA-tail Interacting Protein (MTIP). Here we present one crystal structure that shows three different conformations of Plasmodium MTIP, one of these in complex with the MyoA-tail, which reveal major conformational changes in the C-terminal domain of MTIP upon binding the MyoA-tail helix, thereby creating several hydrophobic pockets in MTIP that are the recipients of key hydrophobic side chains of MyoA. Because we also show that the MyoA helix is able to block parasite growth, this provides avenues for designing antimalarials.     

Characterization of events preceding the release of malaria parasite from the host red blood cell

The process of merozoite release involves proteolysis of both the parasitophorous vacuole membrane (PVM) and red blood cell membrane (RBCM), but the precise temporal sequence remains controversial. Using immunofluorescence microscopy and Western blotting of parasite-infected RBCs, we observed that the intraerythrocytic parasite was enclosed in a continuous ring of PVM at early stages of parasite development while at the segmented schizont stage, the PVM appeared to be integrated in the cluster of newly formed merozoites. Subsequently, such clusters were detected extraerythrocytically together with single merozoites devoid of the PVM at low frequency, suggesting a primary rupture of RBCM, followed by PVM rupture and release of invasive merozoites. Secondly, since cysteine proteases are implicated in the process of parasite release, antimalarial effects of 4 cysteine protease inhibitors (leupeptin, E64, E64d, and MDL) were tested at the late schizont stage and correlated with the integrity of PVM and RBCM. We observed that leupeptin and E64 treatment produced extraerythrocytic clusters of merozoites associated with PVM suggesting inhibition of PVM lysis but not RBCM lysis. Merozoites in these clusters developed into rings upon removal of the inhibitors. In contrast, E64d and MDL caused an irreversible parasite death blocking further development. Future characterization of the mechanism(s) of inhibition may facilitate the design of novel antimalarial inhibitors.     

Multiple stiffening effects of nanoscale knobs on human red blood cells infected with Plasmodium falciparum malaria parasite

During its asexual development within the red blood cell (RBC), Plasmodium falciparum (Pf), the most virulent human malaria parasite, exports proteins that modify the host RBC membrane. The attendant increase in cell stiffness and cytoadherence leads to sequestration of infected RBCs in microvasculature, which enables the parasite to evade the spleen, and leads to organ dysfunction in severe cases of malaria. Despite progress in understanding malaria pathogenesis, the molecular mechanisms responsible for the dramatic loss of deformability of Pf-infected RBCs have remained elusive. By recourse to a coarse-grained (CG) model that captures the molecular structures of Pf-infected RBC membrane, here we show that nanoscale surface protrusions, known as “knobs,” introduce multiple stiffening mechanisms through composite strengthening, strain hardening, and knob density-dependent vertical coupling. On one hand, the knobs act as structural strengtheners for the spectrin network; on the other, the presence of knobs results in strain inhomogeneity in the spectrin network with elevated shear strain in the knob-free regions, which, given its strain-hardening property, effectively stiffens the network. From the trophozoite to the schizont stage that ensues within 24–48 h of parasite invasion into the RBC, the rise in the knob density results in the increased number of vertical constraints between the spectrin network and the lipid bilayer, which further stiffens the membrane. The shear moduli of Pf-infected RBCs predicted by the CG model at different stages of parasite maturation are in agreement with experimental results. In addition to providing a fundamental understanding of the stiffening mechanisms of Pf-infected RBCs, our simulation results suggest potential targets for antimalarial therapies.The most virulent human malaria parasite, Plasmodium falciparum (Pf), causes ∼700,000 deaths each year (1, 2). Following entry into red blood cells (RBCs), the parasite matures through the ring (0–24 h), trophozoite (24–36 h), and schizont stages (40–48 h). This intraerythrocyte maturation is accompanied by striking changes in the surface topography and membrane architecture of the infected RBC (3–5). A notable modification is the formation of nanoscale protrusions, commonly known as knobs, at the RBC surface during the second half (24–48 h) of the asexual cycle. These protrusions mainly comprise the knob-associated histidine-rich protein (KAHRP) and the membrane-embedded cytoadherence protein, Pf-erythrocyte membrane protein 1 (PfEMP1). KAHRP binds to the fourth repeat unit of the spectrin α-chain, to ankyrin, to spectrin–actin–protein 4.1 complexes, and to the cytoplasmic domain of PfEMP1 (6–9). These attachments enhance the vertical coupling between the lipid bilayer and the spectrin network. Another striking modification in the Pf-infected RBC membrane is the reorganization of the cytoskeletal network caused by parasite-induced actin remodeling (10). As a result of these molecular-level modifications, the Pf-infected RBC exhibits markedly increased stiffness [the shear modulus increases on average from ∼4−10 µN/m in normal/uninfected RBCs, to ∼40 µN/m at the trophozoite stage, and to as high as 90 µN/m at the schizont stage (11–13)] and cytoadherence to the vascular endothelium, which enable sequestration from circulation in vasculature, and evasion from the surveillance mechanisms of the spleen. Although in vitro experimental studies have revealed roles of particular parasite-encoded proteins in remodeling the host RBC (14–22), the mechanism by which Pf-infected RBCs gain dramatically increased stiffness has remained unclear. Indeed, uncertainty remains as to whether the loss of deformability arises from the structural reorganization of the host membrane components or from the deposition of parasite proteins. That is, it is not clear whether the stiffening is due to remodeling of the spectrin network, or to the formation of the knobs, or both. As experimental studies alone have heretofore not been able to determine the molecular details, numerical modeling, combined with a variety of experimental observations and measurements, offers an alternative approach to reveal the underlying mechanisms.We present here a coarse-grained (CG) molecular dynamics (MD) RBC membrane model to correlate structural modifications at the molecular ultrastructure level with the shear responses of the Pf-infected RBC membrane, focusing on the second half of the parasite’s intra-RBC asexual cycle (24–48 h), i.e., the trophozoite and schizont stages. The CG model is computationally efficient, and able to capture the molecular structures of the RBC membrane in both normal and infected states. CGMD simulations reveal that spectrin network remodeling accounts for a relatively small change in shear modulus. Instead, the knobs stiffen the membrane by multiple mechanisms, including composite strengthening, strain hardening, and knob density-dependent vertical coupling. Our findings provide molecular-level understanding of the stiffening mechanisms operating in Pf-infected RBCs and shed light on the pathogenesis of falciparum malaria.     

Aldolase provides an unusual binding site for thrombospondin-related anonymous protein in the invasion machinery of the malaria parasite

An actomyosin motor located underneath the plasma membrane drives motility and host-cell invasion of apicomplexan parasites such as Plasmodium falciparum and Plasmodium vivax, the causative agents of malaria. Aldolase connects the motor actin filaments to transmembrane adhesive proteins of the thrombospondin-related anonymous protein (TRAP) family and transduces the motor force across the parasite surface. The TRAP-aldolase interaction is a distinctive and critical trait of host hepatocyte invasion by Plasmodium sporozoites, with a likely similar interaction crucial for erythrocyte invasion by merozoites. Here, we describe 2.4-A and 2.7-A structures of P. falciparum aldolase (PfAldo) obtained from crystals grown in the presence of the C-terminal hexapeptide of TRAP from Plasmodium berghei. The indole ring of the critical penultimate Trp-residue of TRAP fits snugly into a newly formed hydrophobic pocket, which is exclusively delimited by hydrophilic residues: two arginines, one glutamate, and one glutamine. Comparison with the unliganded PfAldo structure shows that the two arginines adopt new side-chain rotamers, whereas a 25-residue subdomain, forming a helix-loop-helix unit, shifts upon binding the TRAP-tail. The structural data are in agreement with decreased TRAP binding after mutagenesis of PfAldo residues in and near the induced TRAP-binding pocket. Remarkably, the TRAP- and actin-binding sites of PfAldo seem to overlap, suggesting that both the plasticity of the aldolase active-site region and the multimeric nature of the enzyme are crucial for its intriguing nonenzymatic function in the invasion machinery of the malaria parasite.     

Evolution of malaria parasite plastid targeting sequences

The transfer of genes from an endosymbiont to its host typically requires acquisition of targeting signals by the gene product to ensure its return to the endosymbiont for function. Many hundreds of plastid-derived genes must have acquired transit peptides for successful relocation to the nucleus. Here, we explore potential evolutionary origins of plastid transit peptides in the malaria parasite Plasmodium falciparum. We show that exons of the P. falciparum genome could serve as transit peptides after exon shuffling. We further demonstrate that numerous randomized peptides and even whimsical sequences based on English words can also function as transit peptides in vivo. Thus, facile acquisition of transit peptides from existing sequence likely expedited endosymbiont integration through intracellular gene transfer.     

Antibodies to a merozoite surface protein promote multiple invasion of red blood cells by malaria parasites

The 40-50 kDa merozoite surface antigen (MSA2) is a candidate molecule for use in a malaria vaccine. The gene for MSA2 from the 3D7 isolate of Plasmodium falciparum was amplified by polymerase chain reaction and cloned into the bacterial expression vector pGEX-3X to obtain a fusion protein of MSA2 with Schistosoma japonicum glutathione S-transferase. The recombinant fusion protein was used to immunize rabbits. After four injections, the sera had Western blotting and immunofluorescence titres of 10(-6). Immune sera, and immunoglobulin (Ig)G, F(ab)'2, F(ab) prepared from the immune sera, were assessed for their effects on the growth of 3D7 parasites in vitro by microscopy and a [3H]-hypoxanthine incorporation assay. The antibodies did not significantly inhibit red blood cell invasion and parasite growth when added to cultures as 10% v/v serum or as immunoglobulin preparations at concentrations up to 200 microg ml(-1). However, in the presence of IgG or F(ab)'2, but not F(ab), antibodies to MSA2, the proportions of red blood cells invaded by more than one merozoite increased significantly. Multiple invasion is attributed to merozoites cross-linked by bivalent antibodies, attaching to and subsequently invading the same red cell. These observations have a bearing on the evasion of host immune responses by the parasite and the use of full-length recombinant MSA2 protein in a malaria vaccine.