BIOLOGY OF MALARIA SPOROZOITES IN THE MOSQUITO VECTOR

拟议研究的长期目标是了解疟疾子孢子通过蚊子血腔和唾液腺的入侵，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中一个很少研究但至关重要的时期。 破坏腺体侵入所必需的基本受体-配体结合可能是一种基于病媒控制疟疾传播的新方法。 具体目标是：1）分子表征子孢子入侵的候选唾液腺受体。 我们有25个杂交瘤细胞系，产生的单克隆抗体（单克隆抗体）的分子上的埃及伊蚊唾液腺的表面。 几种单克隆抗体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克隆抗体所识别的分子，克隆其同源cDNA，并针对重组蛋白产生多克隆抗体。 2)通过体内侵袭阻断试验测试候选子孢子受体的生物学功能。 将纯化的子孢子与针对候选受体的多克隆抗体一起沿着接种到未感染蚊子的血腔中。 相对于对照，特异性抗体对子孢子侵入的抑制将鉴定子孢子侵入发生所必需的假定受体分子。 3)确定蚊子血腔中未侵入子孢子的命运。 我们的初步结果表明，不侵入唾液腺的子孢子可能在血腔中被破坏。确定子孢子破坏位点是了解潜在特异性免疫应答的细胞和分子基础并将其用于疟疾传播控制的第一步。 我们已经建立了一个基因转化的伯氏疟原虫生产荧光子孢子。 我们将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体内的荧光子孢子，以确定子孢子破坏的部位和细胞机制。 总的来说，我们提出，有一个竞争的子孢子破坏和腺体入侵的过程之间的血腔。 通过阻止腺体侵入和/或增强破坏，最终可能操纵这种动态状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.