BIOLOGY OF MALARIA SPOROZOITES IN THE MOSQUITO VECTOR

这项拟议研究的长期目标是了解疟疾孢子虫通过蚊子血和入侵唾液腺的途径，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研究很少但却至关重要的时期。破坏腺体入侵所必需的受体-配体结合可能是一种基于媒介控制疟疾传播的新方法。具体目的是：1)分子表征候选唾液腺受体的孢子子入侵。我们有25个杂交瘤系，可以产生针对埃及伊蚊唾液腺表面分子的单克隆抗体（mab）。几种单抗抑制鸡状疟原虫孢子子侵入腺体，因此是候选受体。我们将鉴定由腺特异性单克隆抗体鉴定的分子，克隆其同源cdna，并建立针对重组蛋白的多克隆抗体。2)通过体内入侵阻断试验检测候选孢子子受体的生物学功能。纯化的孢子子将与针对候选受体的多克隆抗体一起接种到未感染蚊子的血中。与对照相比，特异性抗体对孢子子入侵的抑制将识别出孢子子入侵发生所必需的推定受体分子。3)确定蚊血中未入侵孢子虫的命运。我们的初步结果表明，孢子子不侵入唾液腺，可能在血液中被破坏。确定孢子子破坏的位点是了解潜在特异性免疫反应的细胞和分子基础并在疟疾传播控制中加以利用的第一步。我们已经制造了一系列经过基因改造的伯氏疟原虫，可以产生荧光孢子体。我们将用这些寄生虫感染冈比亚按蚊，然后用共聚焦显微镜跟踪和量化蚊子体内的荧光孢子体，以确定孢子体破坏的位置和细胞机制。总之，我们认为在血腔中存在着孢子破坏和腺体入侵过程之间的竞争。通过阻断腺体入侵和/或加强破坏，最终有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.