Korir Jackson C, Nyakoe Nancy K, Awinda George, Waitumbi John N
Masinde Muliro University of Science and Technology, Kakamega, Kenya.
Walter Reed Project, Kenya Medical Research Institute, Kisumu, Kenya.
PLoS One. 2014 Aug 21;9(8):e105093. doi: 10.1371/journal.pone.0105093. eCollection 2014.
Complement (C) is a crucial part of the innate immune system and becomes over activated during malaria, resulting in depletion of C components, especially those for lectin pathway (LP), thereby compromising the host's innate defense. In this study, involvement of P. falciparum antigens in C activation was investigated.
A highly synchronous culture of the Dd2 clone of P. falciparum was established in a serum free medium. Supernatants harvested from rings, trophozoites and schizonts at various parasite densities were tested for ability to activate C by quantifying amount of C3b deposited on erythrocytes (E). Uninfected sham culture was used as control. Remnants of each C pathway were determined using Wieslab complement System Screenkit (Euro-diagnostica, Sweden). To identify MBL binding antigens of LP, culture supernatants were added to MBL sepharose columns and trapped antigens eluted with increasing concentrations of EDTA (10 mM, 50 mM and 100 mM) and then desalted before being tested for ability to activate C. The EDTA eluate with highest activity was run on a polyacrylamide gel and silver stained proteins analyzed by mass spectroscopy.
Antigens released by P. falciparum growing in culture activated C leading to C3b deposition on E. Maximal activation at 7% parasitemia was associated with schizont stage (36.7%) compared to 22% for rings, 21% for trophozoites and 3% for sham culture. All the three pathways of C were activated, with highest activation being for the alternative pathway (only 6% of C activation potential remained), 65% for classiical and 43% for the LP. Seven MBL binding merozoite proteins were identified by mass spectrometry in the 50 mM EDTA eluate.
MBL binding merozoite adhesins with ability to activate C pathway were identified. The survival advantage for such pronounced C activation is unclear, but opsonisation could facilitate recognition and invasion of E.
补体（C）是固有免疫系统的关键组成部分，在疟疾期间会过度激活，导致补体成分耗竭，尤其是凝集素途径（LP）的成分，从而损害宿主的固有防御。在本研究中，调查了恶性疟原虫抗原在补体激活中的作用。
在无血清培养基中建立恶性疟原虫Dd2克隆的高度同步培养物。从不同寄生虫密度的环状体、滋养体和裂殖体收获的上清液，通过量化沉积在红细胞（E）上的C3b量来测试激活补体的能力。未感染的假培养物用作对照。使用Wieslab补体系统筛选试剂盒（瑞典Euro-diagnostica公司）测定每条补体途径的残余物。为了鉴定LP的甘露糖结合凝集素（MBL）结合抗原，将培养上清液加入MBL琼脂糖柱，用浓度递增的乙二胺四乙酸（EDTA，10 mM、50 mM和100 mM）洗脱捕获的抗原，然后脱盐，再测试其激活补体的能力。将活性最高的EDTA洗脱物在聚丙烯酰胺凝胶上运行，对银染的蛋白质进行质谱分析。
培养中生长的恶性疟原虫释放的抗原激活补体，导致C3b沉积在红细胞上。在7%的寄生虫血症时，最大激活与裂殖体阶段相关（36.7%），而环状体为22%，滋养体为21%，假培养物为3%。补体的所有三条途径均被激活，替代途径的激活程度最高（仅保留6%的补体激活潜能），经典途径为65%，LP为43%。通过质谱在50 mM EDTA洗脱物中鉴定出7种MBL结合裂殖子蛋白。
鉴定出具有激活补体途径能力的MBL结合裂殖子粘附素。这种明显的补体激活所带来的生存优势尚不清楚，但调理作用可能有助于红细胞的识别和入侵。
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