Cheng L W, Kay O, Schneewind O
Department of Microbiology & Immunology, UCLA School of Medicine, University of California, Los Angeles, California 90095, USA.
J Bacteriol. 2001 Sep;183(18):5293-301. doi: 10.1128/JB.183.18.5293-5301.2001.
During infection, Yersinia enterocolitica exports Yop proteins via a type III secretion pathway. Secretion is activated when the environmental concentration of calcium ions is below 100 microM (low-calcium response). Yersiniae lacking yopN (lcrE), yscB, sycN, or tyeA do not inactivate the type III pathway even when the concentration of calcium is above 100 microM (calcium-blind phenotype). Purified YscB and SycN proteins form cytoplasmic complexes that bind a region including amino acids 16 to 100 of YopN, whereas TyeA binds YopN residues 101 to 294. Translational fusion of yopN gene sequences to the 5' end of the npt reporter generates hybrid proteins that are transported by the type III pathway. The signal necessary and sufficient for the type III secretion of hybrid proteins is located within the first 15 codons of yopN. Expression of plasmid-borne yopN, but not of yopN(1-294)-npt, complements the calcium-blind phenotype of yopN mutants. Surprisingly, yopN mutants respond to environmental changes in calcium concentration and secrete YopN(1-294)-Npt in the absence but not in the presence of calcium. tyeA is required for the low-calcium regulation of YopN(1-294)-Npt secretion, whereas sycN and yscB mutants fail to secrete YopN(1-294)-Npt in the presence of calcium. Experiments with yopN-npt fusions identified two other signals that regulate the secretion of YopN. yopN codons 16 to 100 prevent the entry of YopN into the type III pathway, a negative regulatory effect that is overcome by expression of yscB and sycN. The portion of YopN encoded by codons 101 to 294 prevents transport of the polypeptide across the bacterial double membrane envelope in the presence of functional tyeA. These data support a model whereby YopN transport may serve as a regulatory mechanism for the activity of the type III pathway. YscB/SycN binding facilitates the initiation of YopN into the type III pathway, whereas TyeA binding prevents transport of the polypeptide across the bacterial envelope. Changes in the environmental calcium concentration relieve the TyeA-mediated regulation, triggering YopN transport and activating the type III pathway.
在感染过程中，小肠结肠炎耶尔森菌通过III型分泌途径输出Yop蛋白。当环境中钙离子浓度低于100微摩尔时（低钙反应），分泌被激活。缺乏yopN（lcrE）、yscB、sycN或tyeA的耶尔森菌即使在钙离子浓度高于100微摩尔时（钙盲表型）也不会使III型途径失活。纯化的YscB和SycN蛋白形成细胞质复合物，该复合物结合YopN的包括第16至100位氨基酸的区域，而TyeA结合YopN的第101至294位残基。yopN基因序列与npt报告基因5'端的翻译融合产生由III型途径转运的杂交蛋白。杂交蛋白III型分泌所必需且充分的信号位于yopN的前15个密码子内。质粒携带的yopN而非yopN(1 - 294)-npt的表达可补充yopN突变体的钙盲表型。令人惊讶的是，yopN突变体对钙离子浓度的环境变化有反应，在无钙但有钙时不分泌YopN(1 - 294)-Npt。tyeA是YopN(1 - 294)-Npt分泌低钙调节所必需的，而sycN和yscB突变体在有钙时不能分泌YopN(1 - 294)-Npt。用yopN - npt融合体进行的实验确定了另外两个调节YopN分泌的信号。yopN的第16至100位密码子阻止YopN进入III型途径，这是一种负调节作用，可通过yscB和sycN的表达克服。第101至294位密码子编码的YopN部分在有功能性tyeA存在时阻止多肽穿过细菌双膜包膜运输。这些数据支持一个模型，据此YopN运输可能作为III型途径活性的一种调节机制。YscB/SycN结合促进YopN进入III型途径的起始，而TyeA结合阻止多肽穿过细菌包膜运输。环境钙离子浓度的变化解除TyeA介导的调节，触发YopN运输并激活III型途径。
