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.     

Plasmodium vivax: impaired escape of Vk210 phenotype ookinetes from the midgut blood bolus of Anopheles pseudopunctipennis

The site in the midguts of Anopheles pseudopunctipennis where the development of Plasmodium vivax circumsporozoite protein Vk210 phenotype is blocked was investigated, and compared to its development in An. albimanus. Ookinete development was similar in time and numbers within the blood meal bolus of both mosquito species. But, compared to An. pseudopunctipennis, a higher proportion of An. albimanus were infected (P=0.0001) with higher ookinete (P=0.0001) and oocyst numbers (P=0.0001) on their internal and external midgut surfaces, respectively. Ookinetes were located in the peritrophic matrix (PM), but neither inside epithelial cells nor on the haemocoelic midgut surface by transmission electron microscopy in 24h p.i.-An. pseudopunctipennis mosquito samples. In contrast, no parasites were detected in the PM of An. albimanus at this time point. These results suggest that P. vivax Vk210 ookinetes cannot escape from and are destroyed within the midgut lumen of An. pseudopunctipennis.     

The role of programmed cell death in Plasmodium–mosquito interactions

Many host–parasite interactions are regulated in part by the programmed cell death of host cells or the parasite. Here we review evidence suggesting that programmed cell death occurs during the early stages of the development of the malaria parasite in its vector. Zygotes and ookinetes of Plasmodium berghei have been shown to die by programmed cell death (apoptosis) in the midgut lumen of the vector Anopheles stephensi, or whilst developing in vitro. Several morphological markers, indicative of apoptosis, are described and evidence for the involvement of a biochemical pathway involving cysteine proteases discussed in relationship to other protozoan parasites. Malaria infection induces apoptosis in the cells of two mosquito tissues, the midgut and the follicular epithelium. Observations on cell death in both these tissues are reviewed including the role of caspases as effector molecules and the rescue of resorbing follicles resulting from inhibition of caspases. Putative signal molecules that might induce parasite and vector apoptosis are suggested including nitric oxide, reactive nitrogen intermediates, oxygen radicals and endocrine balances. Finally, we suggest that programmed cell death may play a critical role in regulation of infection by the parasite and the host, and contribute to the success or not of parasite establishment and host survival.     

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.     

离体培养鼠疟原虫动合子形成过程的电子显微镜观察

本文报道了用透射电子显微镜观察离体培养的鼠疟原虫配子体到动合子的发育过程。 鼠疟原虫配子的发生是由嗜锇小体趋向配子体表面开始。雌配子体从红细胞中逸出后,嗜锇小体消失。雄配子体微管形成和鞭毛轴丝集合是从红细胞中逸出前出现的。合子转变为动合子由致密内膜及膜下微管形成时开始,继之形成顶端复合物,随着突起增大,表膜复合物逐渐向后延伸,最后包绕整个虫体,即完成动合子的发育。疟原虫生活史第一次核分裂可能发生在动合子形成期间。本文证实了离体培养的动合子与蚊体内发育的动合子结构相同。     

伯氏疟原虫PbPH表达特点和单克隆抗体传播阻断效应研究

制备抗PbPH单克隆抗体(mAb),检测伯氏疟原虫(Plasmodium berghei)PbPH的表达特点和传播阻断能力。主要通过蛋白免疫印迹(Western blot, WB)和间接免疫荧光法(Indirect immunofluoscent assay, IFA),确定PbPH的表达阶段以及抗PbPH单克隆抗体的特异性。1×10~6个P.berghei感染雌性BALB/c小鼠3 d后,尾静脉收集感染小鼠的血液,与鼠源的抗PbPH单克隆抗体/PBS对照,按照不同的稀释倍数(1?5、1?10、1?50)进行混合培养,观察15 min后,伯氏疟原虫配子体出丝中心数及24 h后动合子形成数的变化。WB和IFA检测抗PbPH单克隆抗体可以识别雌雄配子体、雄配子、雌配子/合子、retort和动合子的表面抗原。体外传播阻断实验中,与PBS对照组相比,在加入不同浓度(1?5、1?10)的抗PbPH单克隆抗体培养后,配子体出丝中心数分别减少了41.7%、32.7%,差异具有统计学意义(P0.05);动合子形成数分别减少了45.1%、14.8%,差异具有统计学意义(P0.05)。抗PbPH单克隆抗体可以有效阻断体外配子体出丝中心和动合子的形成,从而影响伯氏疟原虫在蚊体内的进一步发育和继续传播。     

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.     

Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion

Ookinetes are motile invasive stages of the malaria parasite that enter the midgut epithelium of the mosquito vector via an intracellular route. Ookinetes often migrate through multiple adjacent midgut epithelial cells, which subsequently undergo apoptosis/necrosis and are extruded from the midgut epithelium into the midgut lumen. Hundreds of ookinetes may simultaneously invade the midgut epithelium, causing destruction of an appreciable proportion of the total number of midgut epithelial cells. However, there is little evidence that ookinete invasion of the midgut epithelium per se is detrimental to the survival of the mosquito vector implying that efficient mechanisms exist to restore the damaged midgut epithelium following malaria parasite infection. Proliferation and differentiation of precursor stem cells could replace the midgut epithelial cells destroyed and lost as a consequence of ookinete invasion. Although the existence of so-called "regenerative" cells within the mosquito midgut epithelium has long been recognized, there has been no previously published evidence for proliferation/differentiation of these putative precursor midgut epithelial cells in mature adult female mosquitoes. In the current study, examination of Giemsa-stained histological sections from Anopheles stephensi mosquito midguts infected with the human malaria parasite Plasmodium falciparum provided morphological evidence that regenerative cells undergo division and subsequent differentiation into normal columnar midgut epithelial cells. Furthermore, the number of these putatively proliferating/differentiating regenerative cells was significantly higher in P. falciparum-infected compared to uninfected mosquitoes, and was positively correlated with both the level of malaria parasite infection and midgut epithelial cell destruction. The loss of invaded midgut epithelial cells associated with intracellular migration by ookinetes, therefore, appears to trigger, and to be compensated by, proliferative regeneration of the mosquito midgut epithelium.     

The role of Pvs28 in sporozoite development in Anopheles sinensis and its longevity in BALB/c mice

To develop a vivax malaria vaccine for blocking malarial transmission, the ookinete surface protein Pvs28 was cloned from Korean malaria patients using polymerase chain reaction. The Pvs28 gene consists of 726 bp and encodes 241 amino acids. It was subcloned into the expression vector pQE30 and expressed in Escherichia coli. The expressed recombinant protein, rPvs28, has a molecular weight of about 28 kDa in SDS–PAGE analysis. A monoclonal antibody against rPvs28 was produced using BALB/c mice. It inhibited sporozoite development in Anopheles sinensis mosquitoes (n = 81) which is one of the malaria vectors in Korea, with relatively high antibody titer against rPv28 persisting for more than 6 months. These results indicate that rPvs28 induces an immune response in mice that effectively blocks sporozoite development in mosquitoes. Therefore it could be a vaccine candidate for preventing vivax malaria in Korea.     

Plasmodium male development gene-1 (mdv-1) is important for female sexual development and identifies a polarised plasma membrane during zygote development

Successful development of Plasmodium sexual stages is essential for parasite survival, but the genes involved are poorly understood. We ‘knocked out’ the male development gene-1 (mdv-1) locus in Plasmodium berghei and found it to be important in female gametocyte activation. Indirect immunofluoresence assays show MDV-1 has a punctate cytoplasmic distribution in gametocytes. After activation of both females and males, MDV-1 is more peripherally located but in males exclusively it becomes concentrated in a few large foci. In vitro ookinete conversion assays that test the ability of activated female gametocytes to develop into retort stage ookinetes, suggests a complicit role for MDV-1, with the knock-out parasite producing 86% reduction in ookinetes. The retort stage ookinete develops from the zygote by increasing growth of an apical protrusion and MDV-1 locates at the ‘leading’ extracellular apical pole of this protrusion. In the fully developed ookinete MDV-1 is localised to the posterior pole. In vivo, the knock-out parasites demonstrate a phenotype in which there is a 90% reduction of parasite transmission to oocysts in mosquitoes.