Table_2_Serotyping of Toxoplasma gondii Infection Using Peptide Membrane Arrays.XLSX

The intracellular parasite Toxoplasma gondii can cause chronic infections in most warm-blooded animals, including humans. In the USA, strains belonging to four different Toxoplasma clonal lineages (types 1, 2, 3, and 12) are commonly isolated, whereas strains not belonging to these lineages are predominant in other continents such as South America. Strain type plays a pivotal role in determining the severity of Toxoplasma infection. Therefore, it is epidemiologically relevant to develop a non-invasive and inexpensive method for determining the strain type in Toxoplasma infections and to correlate the genotype with disease outcome. Serological typing is based on the fact that many host antibodies are raised against immunodominant parasite proteins that are highly polymorphic between strains. However, current serological assays can only reliably distinguish type 2 from non-type 2 infections. To improve these assays, mouse, rabbit, and human infection serum were reacted against 950 peptides from 62 different polymorphic Toxoplasma proteins by using cellulose membrane peptide arrays. This allowed us to identify the most antigenic peptides and to pinpoint the most relevant polymorphisms that determine strain specificity. Our results confirm the utility of previously described peptides and identify novel peptides that improve and increase the specificity of the assay. In addition, a large number of novel proteins showed potential to be used for Toxoplasma diagnosis. Among these, peptides derived from several rhoptry, dense granule, and surface proteins represented promising candidates that may be used in future experiments to improve Toxoplasma serotyping. Moreover, a redesigned version of the published GRA7 typing peptide performed better and specifically distinguished type 3 from non-type 3 infections in sera from mice, rabbits, and humans.

胞内寄生虫刚地弓形虫（Toxoplasma gondii）可引发多数温血动物（包括人类）的慢性感染。在美国，目前分离得到的菌株多隶属于4种不同的刚地弓形虫克隆谱系（1型、2型、3型及12型）；而在南美洲等其他大陆，非上述谱系的菌株占主导地位。虫株型别是决定刚地弓形虫感染严重程度的关键因素，因此开发一种非侵入式且低成本的刚地弓形虫感染虫株分型方法，并将基因型与疾病转归相关联，具有重要的流行病学意义。血清学分型（Serological typing）的原理基于宿主抗体多靶向识别免疫优势寄生虫蛋白，这类蛋白在不同虫株间存在高度多态性。然而，当前的血清学检测方法仅能可靠区分2型与非2型感染。为优化此类检测方法，本研究利用纤维素膜肽阵列（cellulose membrane peptide arrays），将小鼠、家兔及人类的感染血清与62种不同多态性弓形虫蛋白的950条肽段进行孵育反应。通过该实验，我们得以鉴定出抗原性最强的肽段，并精准定位决定虫株特异性的关键多态性位点。本研究结果证实了此前报道的肽段的应用价值，同时鉴定出可提升并增强检测方法特异性的新型肽段。此外，大量新型蛋白展现出用于刚地弓形虫诊断的潜力。其中，源自多种棒状体蛋白、致密颗粒蛋白及表面蛋白的肽段，有望成为未来实验中优化刚地弓形虫血清分型的候选靶点。此外，经重新设计的已发表GRA7分型肽段（GRA7 typing peptide）表现更优，可在小鼠、家兔及人类的血清样本中特异性区分3型与非3型感染。