Adhesin of Helicobacter pylon : Identification and therapeutic application

幽门螺杆菌(Hp)附着在胃上皮细胞上是维持感染的关键步骤。生物体与上皮细胞的紧密黏附深刻地影响着上皮细胞的功能，导致各种疾病。这种附着是由识别特定受体的称为粘附素的特定细菌蛋白质介导的。在大多数情况下，细胞表面的糖偶联物已被证明是细菌黏附受体。在幽门螺杆菌的病例中，已经报道了几种具有不同受体特异性的假定粘附素。在这个项目中，我们试图识别能够识别硫脂的粘附素。利用两种不同的亲和层析，一种是肝素-琼脂糖亲和层析，另一种是硫酸纤维素亲和层析，从Hp的超声上清液中鉴定出两种候选蛋白。通过在Hp基因组数据中搜索同源序列，从亲和纯化的蛋白质中获得了几个部分氨基酸序列，其中一个被鉴定为热休克蛋白60(HSP)，另一个为功能未知的蛋白质。由于其他研究人员假设HSP是一种在细菌附着的初始阶段起作用的粘附素，因此我们克隆了HSP基因以阐明HSP的结合特异性。这项工作目前正在进行中。其次，我们试图确定沙土鼠的黏附受体的特性，这种黏附受体被认为是一种合适的人胃溃疡和幽门螺杆菌感染诱发的胃癌的动物模型。在这种动物中，与小鼠相比，硫脂在胃粘膜中大量表达，表明在这种动物中看到的较高数量的细菌附着可能是由于受体的丰富。这一结果现已提交发表。为了进一步证实硫脂作为受体的作用，我们克隆了人硫转移酶。硫转移酶在硫脂缺陷细胞中的表达目前正在进行中。

Attachment of Helicobacter pylon (HP) to gastric epithelium is a crucial step for perpetuating the infection. Tight adhesion of the organism to the epithelium profoundly affects the epithelial function causing various diseases. The attachment is mediated by specific bacterial proteins called adhesins which recognize specific receptors. In most cases, cell surface glycoconjugates have been demonstrated as bacterial adhesion receptors. In the case of HP, several putative adhesins with different receptor specificities have been reported. In this project, we tried to identify adhesins which specifically recognize sulfatide. Using two different affimty chromatographies, one with heparin-sepharose and the other with sulfated cellulose, two candidate proteins from sonicated supernatant from HP were identified. Several partial amino acid sequences of the peptides derived from affinity-purified proteins were obtained By searching homologous sequences in the genome data of HP, one was identified as heat shock protein 60 (HSP) and the other as a functionally unknown protein. Since other researchers hypothesized HSP as a putative adhesin acting at the initial step of bacterial attachment, we cloned HSP gene to clarify the binding specificity of HSP. This work is now in progress. Secondary, we tried to characterize the adhesion receptors in Mongolian gerbils which have been known as a suitable animal model of human gastric ulcer and cancer induced by HP infection. In this animal, sulfatides were abundantly expressed in the gastric mucosa compared with mice, indicating the higher number of bacterial attachment seen in this animal may be due to receptor abundance. This result is now submitted for publication. To further confirm the role of sulfatide as a receptor, we have cloned human sulfotransferase. The expression of sulfotransferase in the sulfatide-deficient cells is now in progress.