Glushakova Svetlana, Yin Dan, Gartner Nicole, Zimmerberg Joshua
Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Malar J. 2007 May 21;6:61. doi: 10.1186/1475-2875-6-61.
Intracellular malaria parasites leave their host erythrocytes to infect neighbouring cells after each cycle of asexual replication. No method is currently available for the direct quantification of parasite release.
To quantify parasite release process, human erythrocytes infected with Plasmodium falciparum were injected into sealed chambers at optimal density, where they progressed through the end of the erythrocyte cycle. Each event of parasite release inside the chamber at the site of erythrocyte rupture leaves on the chamber wall a footprint, composed of 1) separated parasites, 2) a digestive vacuole with haemozoin, and 3) fragments of the ruptured membranes. These footprints are stable for hours, allowing precise identification using differential interference contrast (DIC) microscopy. The relative rate of parasite release is defined as the percent of such footprints out of all schizonts injected and incubated into chamber at 37 degrees C for two hours. The method is highly reproducible, easy to perform, and does not require expensive equipment. Additionally, this method allows one to analyse cell and release site morphology, which adds information about the release process and the quality of the culture. The method is used here to show that swelling of schizonts caused by protein-free media inhibits parasite release.
In this study, a novel method is described to count sites of parasite release by microscopy. Besides the direct estimation of parasite release from infected erythrocytes, this method provides a morphological evaluation of normal infected cells approaching the end of the plasmodial life cycle, or pathological forms accumulated as the result of experimental intervention in the parasite release process. One may now accurately estimate the relative parasite release rate at the time of cycle transition, without any obligatory coupling to parasite invasion.
细胞内疟原虫在无性繁殖的每个周期后会离开其宿主红细胞去感染邻近细胞。目前尚无直接定量寄生虫释放的方法。
为了量化寄生虫释放过程，将感染恶性疟原虫的人类红细胞以最佳密度注入密封小室，使其经历红细胞周期结束。在红细胞破裂部位的小室内，每次寄生虫释放事件都会在小室壁上留下一个痕迹，该痕迹由以下部分组成：1）分离的寄生虫；2）含有疟色素的消化泡；3）破裂膜的碎片。这些痕迹在数小时内保持稳定，可通过微分干涉相差（DIC）显微镜精确识别。寄生虫释放的相对速率定义为在37℃下注入并在小室中孵育两小时的所有裂殖体中此类痕迹的百分比。该方法具有高度可重复性，易于操作，且不需要昂贵的设备。此外，该方法还能让人分析细胞和释放部位的形态，从而增加有关释放过程和培养质量的信息。在此使用该方法表明无蛋白培养基引起的裂殖体肿胀会抑制寄生虫释放。
在本研究中，描述了一种通过显微镜计数寄生虫释放部位的新方法。除了直接估计感染红细胞中的寄生虫释放外，该方法还能对接近疟原虫生命周期末期的正常感染细胞或因寄生虫释放过程中的实验干预而积累的病理形式进行形态学评估。现在可以准确估计周期转换时的相对寄生虫释放率，而无需与寄生虫入侵有任何必然联系。
