Invasive phenotype of Trypanosoma cruzi restricted to a population expressing trans-sialidase.
Invasive phenotype of Trypanosoma cruzi restricted to a population expressing trans-sialidase.
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克氏锥虫的侵袭表型仅限于表达反式唾液酸酶的群体。
DOI：
10.1128/iai.64.9.3884-3892.1996
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发表时间：
1996
影响因子：
3.1
通讯作者：
Ming,M
中科院分区：
文献类型：
--
作者：
Pereira,ME;Zhang,K;Gong,Y;Herrera,EM;Ming,M
克氏锥虫表达一种发育调节的反式唾液酸酶，该酶与恰加斯病的发病机制有关。当感染性锥鞭毛体居住在培养细胞的细胞外环境中时，该酶仅限于一小部分（20% 至 30%）寄生虫。转唾液酸酶在这个亚群（称为 TS+）上表达的生物学意义，而不是在大多数（70% 至 80%）形态相似的锥虫（称为 TS-）上表达，其生物学意义尚不清楚。为了确定 TS+ 和 TS- 亚群在克氏锥虫入侵中的作用，我们使用涂有针对转唾液酸酶 C 末端串联重复单元的单克隆抗体的磁珠制备了纯 TS- 和 TS+ 锥虫群体。除去非粘附的 TS- 锥鞭毛体后，通过用转唾液酸酶单克隆抗体的合成肽表位进行特异性洗脱，将 TS+ 锥鞭毛体从珠中分离出来。通过免疫荧光、免疫印迹和唾液酸酶或唾液酸转移酶活性测量来确认 TS+ 和 TS- 表型。 TS+ 锥虫具有高度侵入性，因为它们比未分级的寄生虫更有效地附着、渗透和在培养的哺乳动物细胞中生长。通过观察人抗转唾液酸酶抗体中和感染，强调了转唾液酸酶在侵袭中的关键作用。更重要的是，与 TS+ 寄生虫相比，TS- 寄生虫在入侵上皮细胞和成纤维细胞方面效率极低。此外，将少量外源转唾液酸酶引入非穿透性TS-寄生虫的悬浮液中，将它们转变为与TS+群体难以区分的高度侵入性表型。入侵表型的拯救是克氏锥虫酶特有的，因为细菌和病毒唾液酸酶不会发生这种情况。体外结果在恰加斯病小鼠模型中得到证实，因为 TS- 锥鞭毛体相对无毒力，而 TS+ 锥鞭毛体比普通寄生虫毒性更强。
Trypanosoma cruzi expresses a developmentally regulated trans-sialidase implicated in the pathogenesis of Chagas' disease. On inhabitation of the extracellular milieu of cultured cells by infective trypomastigotes, the enzyme is restricted to a small (20 to 30%) population of parasites. The biological significance of trans-sialidase expression on this subset, termed TS+, and not on the majority (70 to 80%) of morphologically similar trypanosomes, named TS-, is unknown. To determine the roles of the TS+ and TS- subsets in T. cruzi invasion, we prepared pure populations of TS- and TS+ trypanosomes using magnetic beads coated with a monoclonal antibody specific for the tandem repeat unit of the trans-sialidase C terminus. After removal of nonadherent TS- trypomastigotes, the TS+ trypomastigotes were isolated from the beads by specific elusion with a synthetic peptide epitope of the trans-sialidase monoclonal antibody. Confirmation of TS+ and TS- phenotypes was obtained by immunofluorescence, immunoblotting, and sialidase or sialyl transferase activity measurements. The TS+ trypanosomes were highly invasive, as they attached to, penetrated, and thrived in cultured mammalian cells much more efficiently than did unfractionated parasites. The critical role of the trans-sialidase in invasion was underscored by the observation that infection was neutralized by human antibodies to transsialidase. What's more, the TS- parasites, in sharp contrast to their TS+ counterparts, were extremely inefficient in invading epithelial cells and fibroblasts. Further, introduction of small amounts of exogenous trans-sialidase into suspensions of nonpenetrating TS- parasites converted them to a highly invasive phenotype indistinguishable from that of the TS+ population. Rescue of the invasive phenotype was specific for the T. cruzi enzyme, for it didn't happen with bacterial and viral sialidases. The in vitro results were confirmed in the murine model of Chagas' disease, as TS- trypomastigotes were relatively avirulent while TS+ trypomastigotes were more virulent than unfractionated parasites.