Dallas S D, Rolfe R D
Department of Microbiology and Immunology, School of Medicine, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
J Med Microbiol. 1998 Oct;47(10):879-88. doi: 10.1099/00222615-47-10-879.
Toxigenic Clostridium difficile is isolated from a majority of healthy human infants. The exact mechanism of asymptomatic colonisation is unclear; however, previous studies in this laboratory have shown that components of both the immunoglobulin and non-immunoglobulin fractions of human milk bind to toxin A and prevent its interaction with hamster intestinal brush border membranes (BBMs). Secretory IgA (sIgA) is the primary immunoglobulin found in human milk. As sIgA resists digestion in the infant stomach and passes at high levels into the colon, its ability to bind toxin A was the subject of this investigation. Purified sIgA in concentrations at and below those found in human milk inhibited the binding of toxin A to purified BBM receptors. Heating sIgA to 100 degrees C for 5 min did not affect its inhibitory activity. IgM, IgG and serum IgA did not appreciably inhibit the binding of toxin A to BBM receptors. SDS-PAGE separated sIgA into three major bands: secretory component, heavy chains and light chains. Autoradiography with radiolabelled toxin A revealed that toxin A bound to the secretory component (SC) of sIgA. When the three purified subunits of sIgA were coated on to microtitration wells, SC bound significantly more toxin A than the heavy or light chains of sIgA. Purified SC also inhibited toxin binding to receptors in a dose-dependent fashion similar to sIgA. The heavy and light chains of sIgA did not inhibit toxin A receptor binding. Removing carbohydrates from sIgA and SC by enzymic digestion showed that toxin A binds much less to deglycosylated SC than to glycosylated SC. These data suggest that SC in human milk binds to toxin A and may function as a receptor analogue, protecting human infants against C. difficile-associated disease.
在大多数健康人类婴儿体内都能分离出产毒素艰难梭菌。无症状定植的确切机制尚不清楚；然而，本实验室先前的研究表明，人乳中免疫球蛋白和非免疫球蛋白部分的成分均可与毒素A结合，并阻止其与仓鼠肠刷状缘膜（BBM）相互作用。分泌型IgA（sIgA）是人乳中发现的主要免疫球蛋白。由于sIgA可抵抗婴儿胃内的消化作用，并以较高水平进入结肠，因此其结合毒素A的能力成为本研究的主题。浓度等于或低于人乳中浓度的纯化sIgA可抑制毒素A与纯化的BBM受体的结合。将sIgA加热至100摄氏度5分钟不会影响其抑制活性。IgM、IgG和血清IgA对毒素A与BBM受体结合的抑制作用不明显。SDS-PAGE将sIgA分离为三条主要条带：分泌成分、重链和轻链。用放射性标记的毒素A进行放射自显影显示，毒素A与sIgA的分泌成分（SC）结合。当将sIgA的三个纯化亚基包被在微量滴定孔上时，SC结合的毒素A明显多于sIgA的重链或轻链。纯化的SC也以类似于sIgA的剂量依赖性方式抑制毒素与受体的结合。sIgA的重链和轻链不抑制毒素A与受体的结合。通过酶消化去除sIgA和SC上的碳水化合物表明，毒素A与去糖基化SC的结合远少于与糖基化SC的结合。这些数据表明，人乳中的SC与毒素A结合，可能作为受体类似物发挥作用，保护人类婴儿免受艰难梭菌相关疾病的侵害。
