Channing Laboratory, Brigham and Women's Hospital, and Harvard Medical School, Boston, MA 02115, USA
Proc Natl Acad Sci U S A. 2010 Mar 2;107(9):4359-64. doi: 10.1073/pnas.0915164107. Epub 2010 Feb 16.
Cholera is a severe diarrheal disease caused by the motile Gram-negative rod Vibrio cholerae. Live-attenuated V. cholerae vaccines harboring deletions of the genes encoding cholera toxin have great promise for reducing the global burden of cholera. However, development of live vaccines has been hampered by the tendency of such strains to induce noncholeric reactogenic diarrhea in human subjects. The molecular bases of reactogenicity are unknown, but it has been speculated that reactogenic diarrhea is a response to V. cholerae's flagellum and/or the motility that it enables. Here, we used an infant rabbit model of reactogenicity to determine what V. cholerae factors trigger this response. We found that V. cholerae ctx mutants that produced flagellins induced diarrhea, regardless of whether the proteins were assembled into a flagellum or whether the flagellum was functional. In contrast, approximately 90% of rabbits infected with V. cholerae lacking all five flagellin-encoding genes did not develop diarrhea. Thus, flagellin production, independent of flagellum assembly or motility, is sufficient for reactogenicity. The intestinal colonization and intraintestinal localization of the nonreactogenic flagellin-deficient strain were indistinguishable from those of a flagellated motile strain; however, the flagellin-deficient strain stimulated fewer mRNA transcripts coding for proinflammatory cytokines in the intestine. Thus, reactogenic diarrhea may be a consequence of an innate host inflammatory response to V. cholerae flagellins. Our results suggest a simple genetic blueprint for engineering defined nonreactogenic live-attenuated V. cholerae vaccine strains.
霍乱是一种由运动性革兰氏阴性杆菌霍乱弧菌引起的严重腹泻病。携带缺失编码霍乱毒素基因的减毒霍乱弧菌疫苗有很大希望减轻全球霍乱负担。然而，活疫苗的开发受到这些菌株在人类受试者中引起非霍乱性致腹泻的趋势的阻碍。致腹泻性的分子基础尚不清楚，但有人推测致腹泻性是对霍乱弧菌鞭毛及其所产生的运动性的反应。在这里，我们使用致腹泻性婴儿兔模型来确定哪些霍乱弧菌因素引发了这种反应。我们发现，即使蛋白质没有组装成鞭毛或鞭毛没有功能，产生鞭毛蛋白的霍乱弧菌 ctx 突变体也会引起腹泻。相比之下，约 90%感染缺乏所有五个鞭毛编码基因的霍乱弧菌的兔子没有出现腹泻。因此，鞭毛蛋白的产生，独立于鞭毛的组装或运动性，足以引起致腹泻性。非致腹泻性无鞭毛缺陷株的肠道定植和肠道内定位与有鞭毛的运动性菌株没有区别；然而，无鞭毛缺陷株在肠道中刺激产生的促炎细胞因子的 mRNA 转录本较少。因此，致腹泻性腹泻可能是宿主对霍乱弧菌鞭毛的固有炎症反应的结果。我们的结果表明，用于工程设计定义明确的非致腹泻性减毒霍乱弧菌活疫苗株的简单遗传蓝图。
