Keller Nadine, Naguleswaran Arunasalam, Cannas Angela, Vonlaufen Nathalie, Bienz Marianne, Björkman Camilla, Bohne Wolfgang, Hemphill Andrew
Institute of Parasitology, University of Berne, CH-3012 Bern, Switzerland.
Infect Immun. 2002 Jun;70(6):3187-98. doi: 10.1128/IAI.70.6.3187-3198.2002.
The invasive stages of apicomplexan parasites enter their host cells through mechanisms which are largely conserved throughout the phylum. Host cell invasion is divided into two distinct events, namely, adhesion onto the host cell surface and the actual host cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the host cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in Neospora caninum tachyzoites which, due to its homology to MIC1 in Toxoplasma gondii (TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in Escherichia coli, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37 degrees C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero cells, as well as to Vero cell monolayers. Removal of glycosaminoglycans from the host cell surface and modulation of host cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the host cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.
顶复门寄生虫的侵入阶段通过在整个门中基本保守的机制进入宿主细胞。宿主细胞入侵分为两个不同的事件，即粘附到宿主细胞表面和实际的宿主细胞进入过程。前者主要通过微线体蛋白介导，这些蛋白在与宿主细胞表面建立接触时分泌。到目前为止鉴定出的许多微线体蛋白都含有粘附结构域。我们在此展示了编码犬新孢子虫速殖子中一种460个氨基酸（aa）的微线体蛋白的基因组和相应的cDNA序列，由于其与刚地弓形虫（TgMIC1）中的MIC1具有同源性，被命名为NcMIC1。推导的NcMIC1多肽序列包含一个20个aa的N端信号肽，随后是分别由48和44个aa组成的两个串联内部重复序列。每个重复序列中都整合了一个CXXXCG序列基序，让人联想到血小板反应蛋白相关的粘附蛋白家族。这个基序的定位在TgMIC1和NcMIC1中严格保守。由278个aa组成的C端部分在大肠杆菌中表达，通过免疫荧光和速殖子的免疫金透射电子显微镜，利用在重组NcMIC1上亲和纯化的抗体来确认其在微线体中的定位。对感染组织包囊的小鼠脑进行免疫组织化学分析表明，该蛋白在缓殖子阶段的表达降低。将温度升高到37摄氏度引发分泌后，NcMIC1被释放到培养基上清液中。NcMIC1与胰蛋白酶处理过的圆形Vero细胞以及Vero细胞单层结合。从宿主细胞表面去除糖胺聚糖并调节宿主细胞表面糖胺聚糖的硫酸化显著降低了NcMIC1与宿主细胞表面的结合。使用特定糖胺聚糖的固相结合试验证实NcMIC1与硫酸化糖胺聚糖结合。
