BIOLOGY OF MALARIA SPOROZOITES IN THE MOSQUITO VECTOR

这项拟议研究的长期目标是了解疟疾子孢子通过蚊子血囊的传播和唾液腺的入侵，这是寄生虫传播到新宿主所必需的。这一时期仍然是疟疾生命周期的一个研究很少但很关键的间隔期。破坏腺体入侵所必需的受体-配体结合可能是以媒介为基础的疟疾传播控制的一种新方法。本研究的具体目的是：1)从分子水平研究与子孢子侵袭相关的唾液腺受体。我们有25株杂交瘤株能产生针对埃及伊蚊唾液腺表面分子的单抗。一些单抗可以抑制鸡疟原虫子孢子侵入腺体，因此是候选受体。我们将鉴定腺特异性单抗所识别的分子，克隆它们的同源cDNA，并制备针对重组蛋白的多克隆抗体。2)通过体内侵袭阻断试验检测候选子孢子受体的生物学功能。纯化的子孢子将与针对候选受体的多克隆抗体一起接种到未受感染的蚊子的血腔中。相对于对照，特定抗体对子孢子入侵的抑制将识别发生子孢子入侵所必需的假定受体分子。3)确定未侵入的子孢子在蚊子血腔中的命运。我们的初步结果表明，没有侵入唾液腺的子孢子很可能在血腔中被破坏。确定子孢子破坏的位置是了解潜在特异性免疫反应的细胞和分子基础并将其用于疟疾传播控制的第一步。我们已经建立了一株伯氏疟原虫的转基因品系，以产生荧光子孢子。我们将用这些寄生虫感染冈比亚按蚊，然后通过共聚焦显微镜跟踪和定量蚊子中的荧光子孢子，以确定子孢子破坏的位置和细胞机制。总体而言，我们认为在血腔中，子孢子破坏和腺体入侵的过程之间存在竞争。通过阻止腺体入侵和/或加强破坏，最终可能操纵这种动态以减少疟疾的自然传播。

The long-term goal of the proposed research is to understand malaria sporozoite passage through the mosquito hemocoel and invasion of the salivary glands, which are required for parasite transmission to a new host. This period remains a little-studied but critical interval of the malaria life cycle. Disrupting the essential receptor-ligand binding necessary for gland invasion may be a new approach for vector-based control of malaria transmission. The specific aims are to: 1) Molecularly characterize candidate salivary gland receptors for sporozoite invasion. We have 25 hybridoma lines that produce monoclonal antibodies (mAbs) specific for molecules on the surface of Aedes aegypti salivary glands. Several mAbs inhibit Plasmodium gallinaceum sporozoite invasion into glands, and thus are candidate receptors. We will identify the molecules identified by the gland-specific mAbs, clone their cognate cDNAs, and raise polyclonal antibodies against recombinant protein. 2) Test the biological function of candidate sporozoite receptors by an in vivo assay for invasion blocking. Purified sporozoites will be inoculated into the hemocoel of uninfected mosquitoes, along with polyclonal antibodies directed against candidate receptors. The inhibition of sporozoite invasion by specific antibodies relative to controls will identify presumptive receptor molecules that are necessary for sporozoite invasion to occur. 3) Determine the fate of non-invaded sporozoites in the mosquito hemocoel. Our preliminary results indicate that sporozoites that do not invade the salivary glands, are probably destroyed in the hemocoel. Identifying the site of sporozoite destruction is a first step to understanding the cellular and molecular basis of a potential specific immune response and exploiting it in malaria transmission control. We have made a line of Plasmodium berghei genetically transformed to produce fluorescent sporozoites. We will infect Anopheles gambiae with these parasites and will then track and quantify the fluorescent sporozoites in the mosquitoes by confocal microscopy to identify the sites and cellular mechanism of sporozoite destruction. Overall, we propose that there is a competition in the hemocoel between the processes of sporozoite destruction and gland invasion. By blocking gland invasion and/or enhancing destruction, it may ultimately be possible to manipulate this dynamic state to decrease natural malaria transmission.