Chuenkova M, Pereira M E
New England Medical Center, Department of Medicine, Boston, Massachusetts 02111, USA.
J Exp Med. 1995 May 1;181(5):1693-703. doi: 10.1084/jem.181.5.1693.
Trypanosoma cruzi, the etiological agent of Chagas' disease, expresses a trans-sialidase at highest levels in infective trypomastigotes, where it attaches to the plasma membrane by a glycophosphoinositol linkage. Bound enzyme sheds into the extracellular milieu in a soluble form. Experiments performed in vitro suggest that the trans-sialidase participates in several parameters of T. cruzi-host interactions, like cell adhesion and complement resistance. However, the role that membrane-bound and soluble trans-sialidase plays in the infection of mammals is not understood. To begin to study the role the enzyme may play in vivo, T. cruzi trypomastigotes were inoculated subcutaneously into mice that had been sensitized for various times with the purified protein. A single dose of either endogenous or recombinant trans-sialidase injected into the connective tissues of BALB/c mice greatly enhanced parasitemia and mortality. Maximum enhancement was achieved with 1-2-h priming. Injection of the enzyme after the parasites had been established in the inoculation site had little, if any, consequence in modifying virulence. The enhancement did not seem to be through a direct effect of the enzyme on trypomastigote-host cell interactions because it occurred when the sites of trans-sialidase sensitization and parasite inoculation were physically separate. Rather, virulence enhancement seemed to depend on inflammatory cells, since priming with trans-sialidase had no significant effect in severe combined immunodeficiency mice, which lack functional T and B lymphocytes. However, antibody response to T. cruzi in the trans-sialidase-primed BALB/c mice was the same as in the control animals. Virulence enhancement was specific for the trans-sialidase because it did not occur in mice primed with Newcastle virus sialidase, which has the same substrate specificity as the T. cruzi enzyme, or with the sialidase from the bacterium Vibrio cholerae, whose substrate specificity is broader than the trypanosome sialidase. Furthermore, no enhancement of virulence occurred after sensitization with another adhesion protein (penetrin) purified from T. cruzi trypomastigotes and engineered bacteria, nor with bacterial lipopolysaccharide. The virulence-promoting activity of soluble trans-sialidase in the mouse model may be physiologically relevant because it was achieved with tiny doses, approximately 1-2 microgram/kg, raising the possibility that neutralization of the enzyme with specific probes could impair the development of Chagas' disease. In fact, a monoclonal antibody specific for the tandem repeat in the trans-sialidase COOH terminus enhanced infection of BALB/c mice, in agreement with earlier experiments in vitro, whereas antibodies against an amino acid sequence in the Cys region had the opposite effect.
克氏锥虫是恰加斯病的病原体，其感染性锥鞭毛体中反式唾液酸酶的表达水平最高，该酶通过糖基磷脂酰肌醇连接附着于质膜。结合的酶以可溶形式释放到细胞外环境中。体外实验表明，反式唾液酸酶参与克氏锥虫与宿主相互作用的多个参数，如细胞黏附和补体抗性。然而，膜结合型和可溶性反式唾液酸酶在哺乳动物感染中的作用尚不清楚。为了开始研究该酶在体内可能发挥的作用，将克氏锥虫锥鞭毛体皮下接种到用纯化蛋白进行不同时间致敏的小鼠体内。向BALB/c小鼠的结缔组织中注射单剂量的内源性或重组反式唾液酸酶可显著提高寄生虫血症和死亡率。1-2小时的预致敏可实现最大程度的增强。在接种部位已建立寄生虫后注射该酶对改变毒力几乎没有影响（如果有影响的话）。这种增强似乎不是通过该酶对锥鞭毛体与宿主细胞相互作用的直接影响，因为当反式唾液酸酶致敏部位和寄生虫接种部位在物理上分开时也会发生这种增强。相反，毒力增强似乎取决于炎性细胞，因为用反式唾液酸酶预致敏对严重联合免疫缺陷小鼠（缺乏功能性T和B淋巴细胞）没有显著影响。然而，反式唾液酸酶预致敏的BALB/c小鼠对克氏锥虫的抗体反应与对照动物相同。毒力增强对反式唾液酸酶具有特异性，因为在用新城疫病毒唾液酸酶（其底物特异性与克氏锥虫酶相同）或霍乱弧菌唾液酸酶（其底物特异性比锥虫唾液酸酶更广泛）预致敏的小鼠中未发生这种情况。此外，用从克氏锥虫锥鞭毛体和工程细菌中纯化的另一种黏附蛋白（穿膜肽）或细菌脂多糖致敏后，毒力没有增强。小鼠模型中可溶性反式唾液酸酶的毒力促进活性可能具有生理相关性，因为只需微小剂量（约1-2微克/千克）就能实现，这增加了用特异性探针中和该酶可能损害恰加斯病发展的可能性。事实上，针对反式唾液酸酶COOH末端串联重复序列的单克隆抗体增强了BALB/c小鼠的感染，这与早期体外实验一致，而针对Cys区域氨基酸序列的抗体则具有相反的效果。
